8/6/2019 Tailoring cAMP Signalling

1/8

/

T I B S 2 2 - JUNE 199 7

T a i ]o r ia g c A M P o s ig a i]in g r e @ o n s e sthro ag h iso or m ul ti# ]Jo lty

M i le s O . H o u s l a y a n d G r a e m e M it lig a nM u l t i p l e f o r m s o f c A M P p h o s p h o d i e s t e r a s e s ( PD E ), a d e n y l a t e c y c l a s e a n dp r o t e i n k i n a s e A (P K A ) a l l o w c e i l s t o t a ia o r t h e r e s p o n s i v e n e s s o f th e c A M P -s i g n a l l in g s y s t e m a n d t o a ll o w fo r i ts d y n a m i c a d j u s t m e n t . M u l t i p le f o r m s o ft h e s e e n z y m e s c o n f e r s p at ia ~ a n d t e m p o r a l c h a r a c t e r i s t i c s o n c A M P s i g n a lU n gs o a s t o a f f e c t c o m p a r t m e n t a l i s e d r e s p o n s e s w i t h i n a s i n g l e c e l l t y p e . T h ea b i l it y t o b r e a c h t h e P K A a c t i v a t i o n t h r e s h o l d c a n d e p e n d u p o n e i t h e r o r b o t ht h e a c t i v a t i o n o f a d e n y l a t e c y cm a s e a n d i n n i b i t i o n o f s p e c i f i c P D E i s o f o r m s .

O U R K N O W L E D G E O F t h e b r e a d t h o fi n t r a c e l l u l a r s i g n a ll i n g s y s t e m s i n e u k a r y -e r i c c e l l s h a s i n c r e a s e d d r a m a t i c a l l yo v e r t h e p a s t t w o d e c a d e s . I t i s i n t r i -g u in g , h o w e v e r , t h a t w h i l e c A M P s e r v e da s t h e p a r a d i g m f o r t h e s e c o n d m e s s e n -g e r c o n c e p t , o u r a p p r e c i a t i o n of th e c o m -p l e x i ty o f t h e m o l e c u l a r m a c h i n e r y i n -v o l v e d i n i t s g e n e r a t i o n , o b l i t e r a t i o n a n dd e t e c t i o n i s fa r f ro m c o m p l e t e . F e w p e o -p l e r e a l i se t h a t m a m m a l i a n c e l l s h a v e t h eo p t i o n o f s y n t h e s i z i n g u p t o n i n e f o r m so [ a d e n y l a t e c y c l a s e a n d a r o u n d 3 0 fo r m so f e n z y m e s h a v in g c A M P p h o s p h o -d i e s t e r a s e ( PD E ) a c t i v i t y ( T a b le D , a n dt h a t t h e h e t e r o d i m e r i c p r o t e i n k i n a s e A( P K A ) e n c o m p a s s e s m u l t i p l e f o r m s o fb o tl a r e g u l a t o r y a n d c a t a l y t i c u n i ts . A l lt h i s t o p r o d u c e , d e t e c t a n d d e g r a d e as i n g l e s i g n a l l i n g s p e c i e s , n a m e l y c AM P .N e v e r t h e l e s s , s u c h a m o l e c u l e c a n i n fl u -e n c e a b e w i M e r i n g a r r a y o f m e t a b o l i cp r o c e s s e s , i n c l u d i n g g l u c o n e e g e n e s i s ,g l y c o g e n o l y s i s a n d l i p o g e n e s is , a s w e l la s s e c r e t o r y p r o c e s s e s , m u s c l e c o n t r a c -t i on , l e a r n in g , i o n c h a n n e l c o n d u c t a n c e ,p r o - i n f l a m m a t o r y c y t o k i n e p r o d u c t i o na n d a c t i o n , d i f f e r e n t i a t i o n , a p o p t o s i sa n d g r o w t h c o n t r o l , f o r e x a m p l e .M u c h e f f o r t i n a n a l y s i n g s i g n a ll i n g s y s -t e m s h a s b e e n d i r e c t e d t o w a r d s d e t e r -m i n i n g t h e c o m p o n e n t s i n v o l v e d , t h e i rr e g u l a t i o n a n d p o s i t i o n i n s i g n a l t r a n s -d u c t i o n c a s c a d e s . H o w e ve r , v e r y f e w c o n -t r o l s y s t e m s f u n c t i o n e i t h e r i n is o l a t i o no r w i t h o u t r e g a r d t o t h e s p a t i a l lo c a t i o n

o [ t h e p r o t e i n s i n v o l v e d i n s i g n a l g e n -e r a t i o n , d e t e c t i o n a n d t e r m i n a t i o n . T h ei s o [o r m d i v e r s i t y s e e n f o r c o m p o n e n t so f t h e A M P - s i g na l li n g s y s t e m a p p e a r s t ob e i n h e r e n t i n a v a r i e t y o f s ig n a l li n g s y s -t e m s . T h i s i s e v i d e n t f r o m a g r o w i n ga w a r e n e s s o f m u l t i p M s p e c i e s i n v o l v e di n , fo r e x a m p l e , i n o s i t o l p h o s p h a t e d e g r a -d a t i o n , p r o t e i n p h o s p h a t a s e s , k i n a s ec a s c a d e s a n d m i n i - G - pr o t e in s .D e t a i le d u n d e r s t a n d i n g o f t h e f un c -t i o n a l s ig n i f i can ce o f m u l t i p l i c i t y o f cA M P-s ig n a ll in g c o m p o n e n t s h a s y e t t o b e r e -s o l v e d . H o w e ve r , m u l t i p l i d t y a p p e a r st o a l l o w f o r: ( i ) i n t r a c e l l u l a r t a r g e ti n g ;

Enzyme

REVIEWS( 2 ) c r o s s t a | k b e t w e e n a w i d e v a r i e t y( a l l ? ) o f o t h e r s i g n a l l i n g s y s t e m s ; a n d(3 ) ' f i u x -co n tro tR ed sen s i t i v i t y~. i .e. de te r-m in in g w h e th e r " t h e t h r e s h o l d f o r s ig n a lt r a n s d u c t i o n c a n b e b r e a c h e d b y ei t h e rs o l e l y i n c r e a s i n g t h e p r o d u c t i o n o f t h es e c o n d m e s s e n g e r o r , i n a d d i ti o n , b y de -c r e a s i n g i t s d e g r a d a t i o n . I n d i v id u a l c e i lt y p e s a p p e a r t o h a v e t h e p o t e n t i a l t ot a i l o r t h e c h a r a c t e r i s t i c s t h a t d e f i n e b a t ht h e k i n e t i c s o f r e s p o n s i v e n e s s a n d a l s ot h e s p a t i a l o r g a n i s a t i o n w i t h i n t h e c e l lo f t h e i r c A M P - s i g n a l li n g s y s t e m . I n d e e d ,t h e ' c r o s s t a l k ' p o t e n t i a l o f t h e c A M P -s i g n a l li n g s y s t e m , c o u p l e d w i t h s p a t i a la n d t e m p o r a l c o n t r o l , m a y p r o v i d e c e ll sw i t h a ' c o r e ' m e c h a n i s m f o r in t e g r a t i n g /m o d u l a t i n g r e s p o n s e s c o m i n g f r o m av a r i e t y o 1 d i s t i n c t s ~ g n a l li n g s y s t e m s .C o m p o n e n t s o f c A M P s y n t h e s i s a n dd e g r a d a t i o nA d e n y l a t e c y c l a s e s ( A C ) f o r m a l a rg e ,m u l t i - g e n e f a m i l y ( T a b l e 1 ) a n d h a v e ac o m p l e x t r a n s m e m b r a n e t o p o g r a p h y( F i g . 1 ) r e m i n i s c e n t o f m e m b r a n e t r a n s - -p o r t e r s , a l t h o u g h n o s u c h a c t i v i t y h a sy e t b e e n a t t r i b u t e d t o a n y m a m m a l -t a n i s o f o r m s L N e v e r t h e l e s s , o n e i n t r i -g u i n g o b s e r v a t i o n i s t h a t t h e r e c o n s t i t u -t i o n o f Paramecium a d e n y l a t e c y c l a s ei n t o a r t i f ic i a l b i t a y e r s a p p e a r e d t o g e n e r -a t e a v o l t a g e - d e p e n d e n t K c h a n n e l A sw i t h c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r -a s e s , s p l i c e v a r i a n t s o [ a d e n y l a t e c y c l a s e

Tab le L D i f f e r e n t i a l r e g u l a t i o n o f t h e m u l t i p l e f o r m s o f e y l i c n u e | e o t i d ep h e s p h o d i e s t e m s e s a n d e d e n y l a t e e y c i a s e s aRegulators

Sp l ice Chromosom e eGMP on G-pro te in G-pro te invariants Iocaltsat ion Ca~" cAMP [3"y ~-G PKCeA M P -P D EPDEIA 2PDEIBPDEIC 5PDE2 2PDE3A

PDE3BP OE 4A 5PDE4B 3PDE4C 3PDE4D 5PDE7Adenylate cyclaseAC-IAt-I tAC-IIIAC-IVAC-V 2AC-VI 2AC-VIIAC-VIItAC-IX

PKA

M . D . H o u s l a y a n d G . Mi l l i g a n a re i n t h eMo lecu la r Pha rmaco logy Group , D iv i s ion o fB ioche mis t ry and M o lecu la r B io logy , Wo l f sonand Da v idson Bu i ld ing , I ns t i t u te o f L i fe andB iom ed ica l Sc iences , Un ive rs i ty o f G lasgow,G lasgow, Sco t land , UK G12 8QQ.Ema i i : m.hous lay@b io .g la .ac .ukg . m i l l i g a n @ b i o . g l a . a c . u kCopyright 1997, Else vierScienc e Ltd All rights res erved . 0968-0004/97/$17.00

? $ (~) n /a n /a12q13 $ (~) n /a n /a 97 ~ (~) n /a n /a? nc T n /a n /a11p15.1 no ~ n /a n /a ?11p15 nc $ n /a n /a ?19p13.2 ? nc n /a n /alp31 ? nc n /a n /a19p13.1 ? nc n /a n /a 95q12 $ nc n /a n /a~q13-q22 nc nc n /a n /a 9

nc/?nc/?nc/?n cTtnc /?nc /?no/?T(for4D3)?

7p12-7p13 T n /a $ $ T ?5p15.3 nc n /a T(+ ~G ) nc T ?2p22-2p24 T(+ eGs) n /a nc ? ~ ?14q11.2 nc n /a T(+ eGO nc nc /$ ?3q13.2-3q21 $ n /a nc $ nc/Tl~q12 -1 2q13 $ n /a nc $ nc /$16q12-16q13 nc n /a (9) ? (1)? ?8q24.2 T n / a (~) ($} ? ?? ~ n/a nc ? ? ?sT, signif ies increase in act ivity; $, signif ies decrease in act ivity; nc, signif ies no change in act ivity;n/a , s ignif ies not applicable; (+ Gs), signif ies synergism seen with riG . b nhibited by CAMK-IL

Pll: S09684)004(97)01050-5 ~ 'l ~

8/6/2019 Tailoring cAMP Signalling

2/8

REV I EWS( a ) C y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e s

- 3 3 0 r e s i d u e s

T a r g e t i n g R e g u l a t o r y C a t a l y t ic R e g u l a t o r yS e e n f o r . S e e n f o r : S e e n f o r a l l P D E s S e e n f o r:e P O E 4 s p l ic e * P D E 1 o P D E 1v a r i a n t s , i n c l u d e s ( 2 C a M b i n d i n g ; i n h i b i t o r y ) ( s p l i c e v a r i a n t s )S H 3 b i n d in g P D E 2m o t i f ( s ) ( c G M P b i n d i n g ; s t i m u l a t o ry ) PDE3 ( ? ) = P D E 3( P IC A - a n d i n s u l i n - r e g u l a t e dp h o s p h o r y l a t i o n )o P D E 4( 2 U C R r e g i o n s ; P I C Ap h o s p h o w l a t i o n i n 4 D 3 )

( b ) A d e n y l a t e c y c l a s e sF ir st t r a n s r n e m b r a n e c l u s te r S e c o n d t r a n s m e m b r a n e c l u s te r

M 1 M 2J a r ; M a j o r pn s i t e

[~7-,'

UI I IU I I I y I :t l 1 (: ~

F i g u r e 3 .Schemat i c s t ruc tu re o f (a ) cAMP-hydro l ys ing phaspho d ies te rases (POE) and (b } ad eny la te cyc lases .Seque nce ana lys i s and mutage nes is /de le t ion s tud ies o f va r ious c loned PDE orms have a ll owed he iden-t if i ca ti on o f a pu ta t i ve ca ta l y t ic domain . A t e i the r s ide o f th i s a re va r ious func t i ona l doma ins tha t charac-te r ise spec i f i c PDE so fo rms. The pu ta t i ve s t ruc tu re o f a deny la tecyc l a se h a s b e e n d e d u ce d r o m se q u e n ceana lys i s , imp ly ing 12 t ransmem brane he l i ces , and f rom func t i ona l s tud ies tha t l oca te the ca ta l y t i c andvar ious regu la to ry s ites to the i n t race l l u la r cy toso li c - loca ted e g ions o f th e mo lecu le . Recen t l y , he c rys ta ls t ruc tu re o f an eng ineered so lub le a deny la te cyc lase has been d e te rmined 3 , Th is i s c o ,~s i s ten t w i ths tud ies4s nd ica t i ng ha t i n the absence o f rece p to r s t imu la t ion , he ca ta l y ti c ac ti v ity o f th i s t ransm em branee n zyme w a s i n fl u e n ce d b y t h e l i p i d e n v i ro n me n t o f t h e i n k ie r h a l f o f t h e b i la ye r , b u t w h e n co u p l e d t o at ransmem brane recep to r , s t imu la t ion cou ld be f rom the ~ ip idenv i ronmen t o f bo th ha lves o f the b i l ayer .isoforms exist 2, which differ in theirglycosylation and regulatory properties.The two large cytoplasmic loops ofadenylate cyclase (C1 and C2) showhigh homology between family members,whereas the two groups of putativetransmembrane helices are quite distinct.This is by complete contrast to G-protein-coupled receptors, in which it isthe transmembrane elements that arehighly conserved between family mem-bers. Th e cytoplasmic regions of adenyl-ate cyclase provide the active site(s)and their expression, as either a single2 ~

functional chimera or even as two sol-uble portions, allows for the reconstitu-tion of enzyme activityL These solubleconstructs of adenylate cyclase can besynergistically stimulated by Gsa andforskolin and inhibited by adenosine(P-site) analogues. Thus, these solubleconstructs must be self-folding entitiesthat contain distinct, functional do-mains responsible for mediating the ac-tion of each of thes e regulatory ligands.Recently, the crystal structure of theC2 region of AC-IIhas been determined 3 na complex with the activating diterpene

T IB S 2 2 - JUNE 199 7and hypotensive agent, for-skolin Fig. 2). Unlike C1, theC2 region of adenylate cyclaseexhibits catalytic activity whenproduced as a soluble species,although its activity is consid-erably le s s t h a n t h a t o b s e r v e dfor the native enzyme. How-ever, the presen ce of the hom-ologous C1 region, either addedas a soluble species or whenforming a C1-C2 chimera, pro-foundly increases the catalyticactivity o f C2.Structural analyses showthat the monomeric C2 unithas a three-layer c~/~ sandwichstructure. However, in crystalst h e s e C 2 m o n o m e r s i n t e r a c t ina highly ordered fashion toproduce dimers. These have aunique wreath-like structurethat is formed by the headsand tails of the monomersbending towards each otherand becomi ng inter~.:~"aed in a'head-to-tail' fashion. The el-fed of this is to generate a dra-matic central cleft whose frontside is 15 A wide x 15 A deep 25 A long (Fig. 2). This cleftundoubtedly forms the activesite ot adenylate cyclase, asthe cavity is lined with residuesthat have been implicated inmutagenesis studies to beinvolved in ATP binding and,in the case of Asn1025 andArg 1029, show n to be essentialfor catalysis.It is fascinating to note thatat each end of this central cav-ity are two intensely hydropho-bic pocke ts formed by residuessupplied by each participatingmonomer. These provide thebinding site for forskolin(Fig. 2). Therefore, it seemslikely that forskolin promotesthe assembly of the dimericspecies and in so doing, re-engineers thestructure of the catalytic site, thus ef-fecting the potent stimulatory effect ofthis diterpene, which is seen for alladenylate cyclases , except AC-IX. Thevery elegance of this interaction presentsthe tantalising possibility of there beingan 'endogenous , forskolin-like' entity thatcan regulate adenylate cyclase.Divalent metal cations also play animportant role in directly regulating

adenylate cyclase function, with Mn=*serving as a potent activator and Mgz+believed to be involved in mediating

8/6/2019 Tailoring cAMP Signalling

3/8

T ~ SS 2 2 - J U N E 1 9 9 7

p h y s i o l o g i c a l a c t i v a t i o n . T h e s t r u c t u r eo f C 2 s u g g e s t s t h a t a n M e 2 ~ - b i n d i n gs i t e ( s ) l i n e s t h e d i m e r i n t e r l a c e a n d , a sw i t h f o r s k o l i n , t h e d i v M e n t c a t i o n sm i g h t e x e r t t h e i r s t i m u l a t o r y a c t i o n s b ya l t e r i n g t h e c o n f o r m a t i o n o f t h e c a t a l y t i cs i t e b y c h a n g i n g t h e i n t e r a c t i o n b e t w e e nt h e t w o m o n o m e r i c u n i t s.S u c h s t r u c t u r a l s t u d i e s d o n e o n t h ef o r s k oi i n -C 2 c o m p l e x i n d i c a t e t h a t d i m e r -i s a t i o n i s c r u c i a l i n f o r m i n g a n a c t i v eadeny]atec y c l a s e s p e c i e s a n d t h a t t h i si s e x p l o i t e d b y r e g u l a t o r y e n t i ti e s . F o rt h e n a t i v e e n z y m e , i t i s t e m p t i n g t o s u g -g e s t t h a t , b y a n a l o g y , t h e c a t a l y t i c a l l ya c t i v e s p e c i e s i s f or m e d f r o m t h e i n t ra -m o l e c u l a r d i m e r i s a t i o n o f t h e C 2 r e g i o nw i t h i t s h o m o l o g o u s C l r e g i o n . H o w e v e r ,t h e p o s s i b i l i t y o f d i m e r i s a t i o n b e t w e e nd i s t i n c t a d e n y l a t e c y c l a s e m o i e t i e s o c -c u r r i n g u n d e r c e r t a i n s i t u a t i o n s c a n n o tb e r u l e d o u t .T h e c a r b o x y l t e r m i n u s o f a d e n y l a t ec y c l a s e h a s a l s o b e e n i m p l i c a t e d i n A TPb i n d i n g a n d i n m e d i a t i n g c e r t a i n r e g u -l a t o r y a c t i o n s 3A. H o w e v e r , t h e s t r u c t u r eo f t h i s r e g i o n c o u l d n o t b e d e d u c e d f r o mc r y s t a l l o g r a p h y s t u d i e s o n C 2, i m p l y i n gt h a t i t w a s d i s o r d e r e d . O n e p o s s i b i l i t y i st h a t i t m i g h t b e c o m e o r d e r e d o n l y w h e nA TP b i n d s t o t h e c a t a l y t i c s it e , p r o v i d -i n g a ' f l a p ' o v e r t h e s u b s t r a t e . S u c h ap l a s t i c i t y m i g h t t h u s m a k e t h i s r e g i o ns u i t a b l e f o r p r o v i d i n g r e g u l a t o r y i n f l u -e n c e s , a s h a s b e e n i n d i c a t e d b y av a r i e t y o f s t u d i e s .C y c li c n u c le o t i d e p h o s p h o d i e s t e r a s e sa r e a l a r g e m u l t i g e n e f a m i ly5-~ th a t p l a y ap i v o t a l r o l e i n c A M P s i gn a l t e r m i n a t i o na n d i n e s t a b l is h i n g c o m p a r t m e n t a l i s e dr e s p o n s e s ( T a b l e 1). S o m e P DE f o r m ss p e c i f i c a l l y h y d r o l y s e c GM P , s u c h a s t h eP D E 6 e n z y m e s , w h i c h a r e a c t i v a t e d b yt h e G p r o t e i n t r a n s d u c i n s u b s e q u e n t t ol i gh t a c t i v a t i o n o f r h o d o p s i n i n t h e v i s u a ls i g na l t r a n s d u c t i o n s y s t e m . O t h e r s a r es p e c i f i c f o r c A M P , s u c h a s t h e P D E3 , -4a n d - 7 fa m i l ie s , w h e r e a s m e m b e r s o f t h eP D E1 a n d P D E 2 f a m i l i e s a r e p r o m i s c u -o u s a n d c a n h y d r o l y s e b o t h c A M P a n dc G M P . T h e l i v e k n o w n f a m i l i e s o f P D E st h a t c a n h y d r o l y s e c A M P ( P D E I , - 2 , - 3 ,- 4, - 7 ) h a v e a c e n t r a l ' c o r e ' r e g i o n o f - 3 3 0a m i n o a c i d s c o n t a i n i n g t h e a c t i v e s i t e( F i g . 1 ) . H e t e r o g e n e o u s r e g i o n s w i t h i nt h is ' c o r e ' r e g i o n c h a r a c t e r i s e p a r t i c u l a rP DE f a m i l i e s a n d , p r e s u m a b l y , d e f i n es u b s t r a t e s p e c i f i c it y a n d s u s c e p t i b i l i t yt o t h e a c t i o n o f s e l e c t i v e i n h i b i to r s . O u t -s i d e t h i s c o r e r e g io n , t h e p r i m a r y s e -q u e n c e o f t h e P D E i s o e n z y m e s i s v e r yd i f f e r e n t , w i t h m a r k e d h e t e r o g e n e i t yo c c u r r i n g a t t h e a m i n o - a n d c a r b o x y -t e r m i n a l r e g i o n s. T h e g e n e s e n c o d i n g

REVIEWS

F i g u r e 2S t r u c t u r e o f t h e C 2 r e g i o n o f t y p e q l a d e n y [ a t e c y c l a s e ( A C - I I) . T h i s s h o w s t h e c a t a l y t icC2 d imer o f r a t A C - I I w i t h m o l e c u l e s o f f o r s k o l i n b o u n d a t e i t h e r e n d o f t h e c a t a l y t i c s it ef o u n d i n a c l e f t f o r m e d b e t w e e n t h e t w o C 2 u n i t s . T h i s f ig u r e i s r e p r o d u c e d w i t h k i n d p e r-m i s s i on o f .L H . Hu r l ey 3 .

P DE s a p p e a r t o b e h i g h l y c o m p l e x w i th ,f o r e x a m p l e , a t l e a s t 1 6 e x o n s s p a n n i n gt h e 4 9 k b o f s e q u e n c e r e q u i r e d f o r t h ea s s e m b l y o f a h u m a n P DE 4A is o f o r m .P a r t o f t h i s c o m p l e x i t y u n d e r p i n s t h ep r o d u c t i o n o f a c o m p l e x p a t t e r n o f i s o -t o r m s , s e e n f o r t h e P D E i , -2 a n d - 4 g e n e s ,w h i c h a r e g e n e r a t e d b y a l t e r n a t i v em R N A s p l i c i n g a n d e x p r e s s e d i n a c e il -t y p e s p e c i f i c f a s h io n .Rux.e0ntr011edsen s i t i v i t yB a s a l c A M P l e v e l s i n m o s t c e l l s a r e-1 ~ tM, wi t h a lev el o f -10 t~M cAM P pr o -v i d i n g t h e t h r e s h o l d f o r a c t i v a t i o n o fP K A . T h e o r e t i c a l l y , a t l e a s t , t h i s t h r e s h -o l d c a n b e b r e a c h e d e i t h e r b y a c ti v a t io no f a d e n y l a t e c y c l a s e o r b y i n h i b i ti o n o [c A M P- PD E ( F i g . 3) . W h a t i n v a r i a b l y d e -t e r m i n e s w h i c h o f t h e s e t w o p r o c e s s e sc a n p r o v i d e r e g u l a t i o n i n a p a r t i c u l a r c e l lt y p e i s t h e l e v e l o f ' f u ti l e c y c l i n g ' o f c A M Pu n d e r r e s t i n g c o n d i t i o n s . T h i s i s i t se l f d e -t e r m i n e d b y t h e b a s a l a c t i v i t y o f a d e n y l -a t e c y c l a s e a n d t h u s , s p e c i f i c al l y , b y t h ea d e n y l a t e c y c l a s e i s o f o rm s t h a t a r e p r o -d u c e d i n a p a r t i c u l a r c e l l t y pe . F o r e x -a m p l e , A C -I I h a s a s u b s t a n t i a l l y g r e a t e rb a s a l a c t i v i t y t h a n A C -V P. B a s a l a d e n y l -a t e c y c l a s e a c t i v i t y c a n a l s o b e a f f e c t e db y t h e c o n c e n t r a t i o n o f G f c o u p l e d r e c e p -t o r s : h i g h l e v e l s o f t h e s e r e c e p t o r s , s u c h

a s a r e s e e n i n o v e r e x p r e s s i o n s y s t e m s ,c a n a c t i v a t e G i n a n a g o n i s t - i n d e p e n d e n tf a s h i o n , y i e l d i n g a n e n h a n c e d ' b a s a l 'a d e n y l a t e c y c l a s e a c t i v i t y ~a .i n c~ l ls s u c h a s a d J p o c y t e s , t h e b a s a la d e n y l a t e c y c l a s e ~ c t i v i t y i s h i g h , a n dc A M P l e v e l s a r e k e p t b e l o w t h e t h r e s h -o l d f o r P K A a c t i v a t i o n t h r o u g h t h e a c t i v -i t y o f P D E l s o f o r m s g e n e r a t i n g a c y c l i cA M P ' f u t i l e c y c l e ' . T h i s i s a h i g h l y s e n s i -t i v e s y s t e m t h a t a l l o w s f o r t h e b r e a c h -i n g o f t h e P I CA a c t i v a t i o n t h r e s h o l de i t h e r b y s m a l l i n c r e a s e s i n a d e n y l a t ec y c l a s e a c t i v i t y o r b y s m a l l r e d u c t i o n so f P D E a c t i v i t y , p l a c i n g c o n t r o l e q u a l l ya t t h e l e v el o f e i t h e r s y n t h e s i s o r d e g r a -d a t i o n o f c A M P ( ' h i g h c y c l i n g ' ; F i g . 2 ) .B y c o n t r a s t , i n h e p a t o c y t e s , t h e b a s a la d e n y l a t e c y c l a s e a c t i v i t y i s e x t r e m e l yl o w a n d P DE i n h i b it i o n s e r v e s o n l y t oc a u s e a m a r g i n a l r i s e i n c A M P c o n c e n -t r a t i o # ~ .13, w h i c h i s i n s u f f i c i e n t t ob r e a c h t h e a c t i v a t i o n t h r e s h o l d f o r P K A( ' l o w c y c l i n g ' ; F i g. 3 ) . T h u s , i n h e p a t o -c y t e s , P K A a c t i v a t i o n i s d e p e n d e n t u p o na c t i v a ti o n o f a d e n y l a t e c y c l a s e a n d c a n -n o t b e a c h i e v e d t h r o u g h P DE i n h i bi t i o na l o n e . N e v e r t h e l e s s , u n d e r c o n d i t i o n s o fa d e n y l a t e c y c l a s e a c t i v a t i o n i n b o t ht h e s e c e l l t y p e s , e i t h e r i n h i b i t i o n o r a c t i -v a t i o n o f P DE a c t i v i t y c a n p r o f o u n d l ya f f e c t t h e d u r a t i o n o v e r w h i c h t h e P K A

219

8/6/2019 Tailoring cAMP Signalling

4/8

REV IEW Sj ( b ) L o w - c A M P c y c l i n g c o n d i ti o n j

-5 P r o t e i nk i n a s e Aa c t i v a t i o nt h r e s h o l d

Time Timeact!vaEioF~ nh ibit{ on ~ AC ,CtlWJtlOr inhibitionA c , P ~ E - ~ C h a n g e I A C . PD E ~ p~ " n . i v . ~ ~ ~ @ I ib a sa l b a sa l b a s a l ~ b a s a l I a C l n Y Jb a s a l b ~ a l b a s a l ~ b a s a l

PDE- PDEH i g h b a s a l A C : H i g h b a s a l P O E , = S ~ t L us J L o w b d sa ~ ,c ,C :

Rgure 3S c h e m a t i c o f t h e d y n a m i c s o f i n t r a c e l l u la r c A MP me t a b o l i s m t h a t i n fl u e n c e p ro t e i nkinase A (PI~J~)a c t iv a t i o n , d e mo n s t r a t i n g t w o p o s s i b l e e x t r e me s o f b e h a v i o u r o f c A M P s i g .na i l i ng . (a ) I n t he 'h igh -cyc ling' cond i t i on , an adeny la te cyc lase i so fo r m w i th a h igh basa lac t i v i t y is exh ib i t ed . To ensu re t ha t , unde r re s t ing c o ~ : : m : , .~ "-~ '~ o ,:;.e.o~ ~n oa inact ives ta te a h igh ph ospho d ies te ras e (PDE) ac t i v i t y is requ i red . I n t hes e ce l l s , t he PKA ac t i va t iont h r e s h o l d c a n b e b re a c h e d b y e i t h e r a c t i v a ti o n o f a d e n y l a t e c y c t a s e (A C ) o r b y P D Einh ib i t i on , (b ) I n the ' low-cycl ing " con d i t i on , an AC iso fo rm w i th a low basa l a c t i v i ty i s syn -thes ized , and requ i res on ly an equ iva len t PDE ac t i v i t y t o ensu re t ha t , unde r res t ing con -d i t i ons . PKA is kep t in an inac t i ve s ta te . I n thes e ce l l s , t he PEA ac t i va t ion t h resho ld i s un -l i ke ly t o b e b reach ed by PDE inh ib i t i on , becaus e the basa l AC ac t i v i ty i s so low, i t wou ldtake a long t ime to ra i se t he cAMP conce n t ra t ion su i tab ly . Thus , i n t he low-cyc ling cond i t i on .A C a c t i v a ti o n d o m i n a t e s a s t h e me a n s f o r t r i g g e r i n g r e s p o n s e s , a l t h o u g h P D E i n h ib i t io nw o u l d b e e x p e c t e d t o a mp l i f y t h i s . T h e t i me s c a l e o f t h e c h a n g e s i n d i c a t e d i n t h e s c h e ma t i ca re o f te n o f t h e o r d e r o f 2 - 1 0 r a i ns t o a c h i e v e ma x i ma l c A MP re s p o n s e s ,

a c t i v a t i o n t h r e s h o l d i s b r e a c h e d . T h u s ,d e p e n d i n g o n w h i c h l s o f o r m s o f b o t hP DE a n d a d e n y l a t e c y c l a s e a r e s y n t h e -s i z e d i n c e l l s , t h e p h y s i o l o g i c a l c o n s e -q u e n c e s c o n c o m i t a n t u p o n m o d u l a t in gP D E a c t i v i t y c a n b e v e r y d i f f e r e n t i n -d e e d . T h i s i s d e t e r m i n e d b y t h e a c t i v i t ya n d r e g u l a t o r y p r o p e r t i e s o f t h e i s o -f o r m s a n d i s l i k e l y t o b e i n f l u e n c e d b yt h e i r p r e c i s e t h r e e - d i m e n s i o n a l I o c a l i s .a t ion in the cel l (F ig. 4) .I n t m c e l l u l a r t a r g e t i n gT h e c o n c e p t o f c o m p a r t m e n t a l i s a t io no f c A M P s ig n a l l in g h a s b e e n m o o t e d t oe x p l a i n t h e n u m e r o u s i n s t a n c e s w h e r e ,i n a s i n g l e c e l l t y p e , t w o r e c e p t o r s c o u -p l e d t o s ti m u l a t e a d e n y l a t e c y c l a s e ( A C )c o u l d a c h i e v e d i s t i n c t c h a n g e s i n c e l -l u l a r f unc t i on i ng ~4a~. F ur t h e r m or e , va r i -o u s s t u d i e s h a v e n o w i d e n t i f i e d b o t ht h e non- u n i f o r m i n t r ace l lu l a r d i s t r i bu t i ono f c A M P 16 a n d t h e m o l e c u l a r m a c h i n e r yt h a t m i g h t a l l o w t h i s t o b e g e n e r a t e d 1 4Js,22O

t h u s s e r v i n g t o r e i n f o r c e t h e n o t i o n o fc o m p a r t m e n t a l i s a t i o n ( Fig . 4 ). S u c h m a -c h i n e r y t ak e s t h e f o r m o f c o m p o n e n t st h a t a r e p r e c i s e l y t a r g e t e d w i t h i n t h et h r e e - d i m e n s i o n a l m a t r i x o f t h e c e l l.T h e s o u r c e o f c A M P p r o d u c t i o n c a nb e I o c a l i s e d to d i s c r e t e r e g i o n s o f t h ep l a s m a m e m b r a n e i n p o la r c el ls , a s s e e nf or va r i ous G~ -l inked r ecep t o r s an d f o r mso f a d e n y l a t e c y c l a s e . T h e m o l e c u l a rb a s i s t h r o u g h w h i c h t h e l a t e r a l s e g r e -g a t io n o f t h e s e c o m p o n e n t s m i g h t b ea c h i e v e d i s s t i l l u n k n o w n , i t m i g h t , f o re x a m p l e , o r i g i n a t e a s a r e s u l t o f th e p r o -t e i n s y n t h e t i c v e s i c u l a r d e l i v e r y s y s t e mo r b y s u b s e q u e n t r e c r u it m e n t , d e t e r -m i n e d b y p r o t e i n - p r o t e i n i n t er a c ti o n .T h e i n t ra c e l lu l a r d e t e c t o r s y s t e m f o rc A M P i s p r o v i d e d f o r b y P K A ( A - k in a s e ),w h i c h i s a h e t e r o d i m e r i c s p e c i e s c o n -s i s t i n g o f t w o c A M P - b i n d i n g r e g u l a t o r ys u b u n i t s ( R ) a n d t w o p r o t e i n k i n a s ec a t a l y t i c s u b u n i t s ( C ) . T h e r e a r e t w of o r m s o f t h e r e g u l a t o r y s u b u n i t , R - i a n d

T I B S 2 2 - JUNE 1997R - If , wh er e t he P KA- R I co m pl e x i s e s sen*t ia l ly c y t o s o l ic c o m p a r e d w i t h t h e P K A -R il c o m p l e x , w h i c h [ s a l m o s t e x c l u s i v e lyp a r t i c u l a t e - a s s o c i a t e d . T h e i m m o b i l is -a t i o n o f PK A -R II a p p e a r s t o b e m e d i a t e db y i ts i n t e r a c t io n w i t h m e m b e r s o f al a r g e f a m i l y o f A - k i n a s e - a n c h o d n g p r o -t e i ns ( AKAPs). T hes e AK AP s a r e p r o -d u c e d i n a c e l l - s p e c i f i c f a s h i o n a n d a r ef o u n d a s s o c i a t e d w i t h a w i d e a r r a y o fs u b c e l l u l a r s t r u c t u r e s , t h u s d e f i n i n g i n -t r a c e l l u l a r l o c a t i o n o n t h e P K A - R II c o m -p l ex r~ ~ 7 . T he r e f o r e , a s ys t em i s i n p l acet h a t p r o v i d e s f o r d i s t i n c t f o a l d e f i n in gt h e s i t e s o f c A M P p r o d u c t i o n , d e t e c t i o nand degr ada t i on ( F i g . 4 ) .cAMP degca~a t ion by t he PDE sys te~ . I ft h e r e w a s n o d e g r a d a t i o n s y s t e m ~ o rcAM P wi thin cel l s ( i.e . PDE act ivi ty) , theni n t r a c e l iu l a r c A M P le v e l s w o u l d r a p i d l yb e c o m e u n i f o r m . T h u s , a k e y f a c t o r i nt h e g e n e r a t i o n o f a n o n - u n i f o r m i n t r a c el -l u l a r d i s tr i b u t i o n o f c A M P i s t h e d e g r a -d a t i v e s y s t e m p r o v i d e d b y P D E a c ti v it y( F i g . 4 ) . T he ab i l i t y t o r egu l a t e P DE ac -t iv i ty c a n t h e r e f o r e b e e x p e c t e d t o p r o -v i d e a p o w e r f u l w a y o f m a n i p u l a t i n g t h em a g n i t u d e a n d d u r a t i o n o f t h e c A M P re -sp on se by de f i n i ng : ( 1 ) t he a r r a y o f di ff er -e n t ia l ly l o c a l is e d P K A s p e c i e s t h a t c a nb e a c t i v a t e d b y c A M P ; a n d ( 2 ) t h e t e m -por a l na t u r e o f t h is ac t i va t i on .F i ne - t un i ng o f t he con t r o l o f d i s t i nc t l yc o m p a r t m e n t a l i s e d P K A -R l l s p e c i e sc o u l d p o t e n t i a ll y b e a c h i e v e d b y th e t a r -g e t i n g o f s p e c i f i c P D E t y p e s t o d i s t i n c ti n t r ace l l u [ a r l oc a t i ons . C e r t a i u l y , i n va r i -o u s c e ll t y p e s P K A - R il c a n b e s e l e c t i v e l ya c t i v a t e d b y d i f f er e n t G - l i n k e d r e c e p -t o r s I'u '~ JT . t wou l d ap pe a r t ha t t he spa t i a ls e p a r a t i o n o f t h e s i t e s o f s y n t h e s i s , d e -t e c t i o n a n d d e g r a d a t i o n o f cA M P is t h u sach i eved i n many , i f no t a l l , c e l l s , andl e a d s t o t h e e s t a b l i s h m e n t o f s te e p s p a -t ia l gradients t~ ' ,~s . This 'compar tmental i s -a t i o n ' h a s f u n c ti o n a l c o n s e q u e n c e s f o r t h ei n t egr a t i on o f ce l l u la r s i gna l l ing r e sp onse sm e d i a t e d b y cA M P a n d t h e m e a n s b yw h i c h t h e y a r e m o d u l a t e d t h r o u g h t h ea c t i o n o f o t h e r s i g na l li n g s y s t e m s .PD E iso fo rm s are t a rg e t ed . W h i l e i t h a sl o n g b e e n a p p r e c i a t e d t h a t p a r t i c u l a t ea n d c y t o s o l i c f o r m s o f P D E s e x i s t e d , i th a s o n l y r e c e n t ly b e c o m e a p p a r e n t t h a ta l a rg e a n d s o p h i s t i c a t e d s y s t e m f o rg e n e r a t i n g P D E i s o f o r m s t a r g e t e d t o d is -t i n c t i n t r a c e ll u l a r s t r u c t u r e s e x i s ts . T h i si s e x e m p l i f ie d b y s t u d i e s o n t h e c A M P -s p e c i f i c P D E4 e n z y m e f a m i l y ~ . '" . T h ef o u r g e n e s t h a t e n c o d e t h is f a m il y a r ed i s tr i bu t e d o n t h r e e c h r o m o s o m e s a n dp r o d u c e a c o m p l e x a r r a y o f P D E i so -f o r m s by a l t e r n a t i ve mR N A sp li ci ng6 .LT h i s t a k e s t h e f o r m o f 5 ' - d o m a i n s w a p s ,

8/6/2019 Tailoring cAMP Signalling

5/8

T I B S 2 2 - J U N E 1 9 9 7 REVIEW Sl e a d i n g t o a f a m i l y of o v e r ! 5 d i f f e r e n ta c t i v e i s o f o r m s b e i n g p r o d u c e d , e a c h o fw h i c h h a s a d i s t i n c t a m i n o - t e r m i n a l r e -g i on . T h e r e a r e t w o 5 ' - s p li c e j u n c t i o n sp r o d u c i n g ' l o n g ' a n d ' s h o r t ' P DE 4 i s o -f o r m s . A n a l y s e s o [ P DE 4A i s o f o r m s s h o wt h a t a m i n o - t e r m i n a l t r u n c a t i o n , p r o d u c -i n g a c o m m o n ' c o r e " u n i t f o u n d i n a l lP D E 4 A i s o f o r m s , y i e l d s a h i g h l y a c t i v es p e c i e s w h i c h i s s y n t h e s i z e d e x c l u s i v e l yi n t h e c e l l c y t o s o l . T h i s i s b y s t a r k c o n -t r a s t t o f u ll = ! en g th i s o f o r m s , w h i c h s h o ws e v e r e l y d i m i n i s h e d c a t a l y t i c a c t i v i t ya n d c a n b e f o u n d a s s o c i a t e d a t s p e c i f i cs u b c e l l u l a r s i t e s 2.O n e e x a m p l e i s t h e ' s h o r t ' P D E 4 A i s o -f o r m R D 1 , w h i c h h a s a 2 3 - a m i n o a c i da m i n o - t e r m i n a l s p l i c e r e g i o n t h a t c o n -f e r s a b s o l u t e m e m b r a n e a s s o c i a t i o n ~o n t h e s o l u b l e ' c o r e ' P D E4 A, a n d w h i c ha l s o s e r v e s t o a t t e n u a t e t h e c A M P - P D Ec a t a l y t i c a c t i v i t y o f t h i s t s o fo r m .S y n t h e s i z e d n a t i v e l y i n b ra i n , a n d a l s ot r a n s i e n t l y i n C O S c e l l s , R D 1 i s a s s o c i -a t e d p r e d o m i n a n t l y w i t h p l a s m a m e m -b r a n e s , a p p a r e n t l y l o c a l i s e d t o u n d e r -l y in g p u n c t a t e v e s i c u l a r s t r u c t u r e s . Af r a c t i o n i s a l s o | o u n d a s s o c i a t e d w i t ht b G o l g i w h e r e , i n d e e d , R D I i s e x c l u -s i v e l y l o c a l i s e d i n s t a b l y t r a n s f e c t e dh u m a n t h y r o i d c e l l s 22.O n e p o s s i b i f i t y i s th a t R D I a s s o c i a t e sw i t h v e s i c l e s t h a t t r a f f i c b e t w e e n t h eG o l gi a n d p l a s m a m e m b r a n e . C e r t a i nl y ,a l l t h e i n f o r m a t i o n f o r t a r g e t i n g R D I i sc o n t a i n e d w i t h i n i t s a m i n o - t e r m i n a ls p l i c e re g i o n , a s a c h i m e r a f o r m e d b e -t w e e n t h i s r e g i o n a n d t h e n o r m a l l y s o l -u b l e b a c t e r i a l e nz y m e c h l o r a m p h e n i c o l~ c e t y i t r a n s f e r a s e (C A T) s h o w s s i m i l a rt a r g e t in g . A n a l y s i s o f t h e s p l i c e r e g i o no f R D I u s i n g I H- NM R ~ 9 s h o w s i t t o b ef o r m e d f r o m t w o , i n d e p e n d e n t l y f o ld i n g,h e l i c a l r e g i o n s s e p a r a t e d b y a m o b i l eh i n g e ( F i g . 5 ) . W i t h i n t h e s e c o n d h e l i c a lr e g i o n is a c o m p a c t , i n t e n s e l y h y d r o -p h o b i c , t r y p t o p h a n - r i c h d o m a i n , w h i c hp r o v i d e s th e f o c u s f or m e m b r a n e a s s o c i -a t i on . T h is s e e m s m o s t l i k e l y t o b ea c h i e v e d b y e i t h e r d i r e c t o r i n d i r e c ta s s o c i a t i o n w i t h a n a s y e t u n i d e n t i f i e di n t e g r a l m e m b r a n e p r o t e i n . T h e f u n c -t i o n a l r o l e o f s u c h t a r g e t i n g is u n k n o w n .H o w e v e r , o n e p o s s i b i l i t y i s t h a t i t p l a y sa r o l e i n e n d o c y t o t i c / e x o c y t i c e v e n t st h a t a r e i n f l u e n c e d b y c A M P.B y c o n t r a s t , t h e ' l o n g ' P DE 4A i s o f o t m( R P D E 6 ) a n d i t s h u m a n h o m o l o g u e( P D E 4 6 ) a r e f o u n d d i s t r i b u t e d b e t w e e nb o t h t h e c y t o s o l a n d t h e c o r t i c a l c y t o -s k e l e t o n 2 a. S u c h t a rg e t in g i s t h o u g h t 24 tob e a r e s u l t o f i n t e r a c t i o n s o c c u r r i n gb e t w e e n a n c h o r i n g p r o t e i ns h a v i n g S r ch o m o l o g y 3 (S H 3 ) d o m a i n s a n d t h e

IntemaJstructure

PKA-RII

PKA-Ri

@PKA-RIS o m e h a v e i t a n d o t h e r sh a v e n ' t : a t a l e o fc o m p a r t m e n t a l i s e d c A M P

Rgare 4F a c e ts o f t h e c o m p a r t m e n t a l i s a t i o n o f c A M P p r o d u c t i o n , d e t e c t i o n a n d d e s t r u c t i o n . T h e c o r ep l a y e r s in v o l v e d a r e a d e n y l a t e c y c l a s e s ( A C ) in v o l v e d i n c A M P g e n e r a t i o n , c A M P p h o s p h o -d i e s t e r a s e s ( P D E ) in v o l v e d i n c A M P d e g r a d a t i o n , a n d p r o t e i n k i n a s e A ( P K A ) i n v o lv e d i nc A M P d e t e c t i o n . A d e n y l a t e c y c l a s e i s s e e n l o c a t e d a t d e f i n e d r e g i o n s o f t h e p l a s m a m e m -b r a n e o w i n g t o r e s t r i c t io n s o n i t s f r e e l a t e r a l d i f f u s i o n i n th e m e m b r a n e . T h i s m a y i n v o l v ei n t e r a c t io n s w i t h cytoskeletal c o m p o n e n t s . D i f f e r e n t A C is o f o r m s m a y s h o w d i s t i n c t I o c a li s -a t i o n p a t t e r n s , d i f f e r e n t s u s c e p t i b i l i ti e s t o b e s t i m u l a t e d b y v a r i o u s G s -c o up le d r e c e p t o r sa n d , h e n c e , c a n b e r e s p o n s i b l e f o r g e n e r a t i n g d i s t i n c t p a t t e r n s o f P K, a c t i v a t i o n . T h e cA M Pp r o d u c e d b y A C a t t h e p l a s m a m e m b r a n e d i f f u s e s o u t i n t o t h e c y t o s o l w h e r e b o t h a n c h o r e d( c h a in s ) a n d f r e e , c y t o s o l i c P D E a c t i v i ti e s a l l o w g r a d i e n t s o f d i f f e r e n t c A M P c o n c e n t r a t i o n st o b e e s t a b l i s h e d ( b t u e ) . A n c h o r e d P D E s c a n a l s o c r e a t e I o c a l i s e d c A M P g r a d i e n t s .C h a n g e s i n cA M P c o n c e n t r a t i o n s i n s p e c i fi c i n t r a c e l l u l a r c o m p a r t m e n t s ( b l u e s h a d e s ) c a nb e s a m p l e d a n d m o n i t o r e d b y a n c h o r e d P K A -R II i s e f o r m s . B y c o n t r a s t t o t h i s , P K A - R i is o -f o r m s c a n s a m p l e c A M P l e v e l s t h r o u g h o u t th e c e l l c y t o s o l. T h e p a t t e r n o f P K A - R li a c t i v a t i o ni n a c e l l w il l d e p e n d u p o n ( 1 ) t h e s i t e o f c A M P p r o d u c t i o n , ( 2 ) th e r a t e o f c A M P h y d r o l y s i s b ys o l u b l e P D E s, ( 3 ) t h e a c t i v it y o f a n c h o r e d P D E s a n d t h e s p e c i fi c l o c a t i o n o f t h e a n c h o r e dP K A -R II . S p a t i a l ly s e g r e g a t e d P D E s a n d P I 4 A s m a y a l s o u n d e r g o d y n a m i c t r a n s l o c a t i o nw i t h i n t h e c e l l, a s s e e n , f o r e x a m p l e , in t h e c a s e o f c y t o s o l i c P KA , w h i c h c a n t r a f f i c b e t w e e nt h e c y t o s o l a n d n u c l e u s , t h u s a l l o w i n g s p a t i a l l y d i r e c t e d c h a n g e s i n t h e r e s p o n s i v e n e s sa n d a c t i o n o f t h e P F ,~ d e t e c t i o n s y s t e m .p r o l i n e - r i c h , S H 3 - b i n d i n g m o t i f s w i t h i nt h e a m i n o - t e r m i n a l s p l i c e r e g i o n s o f t h e s eP D E 4 A f o r m s . S H 3 d o m a i n s a r e d i s t i n c tg l o b u l a r s t r u c t u r e s f o u n d i n a v a r i e t y o fs i g n a ll i n g a n d c y t o s k e l e t a l p r o t e i n s , a n dc a n d i r e c t t h e f o r m a t i o n o f f u n c t io n a l l yi m p o r t a n t c o m p l e x e s . C e r t a i n l y , R P D E - 6s h o w s s p e c i f i c i t y f o r v a r i o u s S H 3 d o -m a i n s a n d c a n b e c o 4 m m u n o p r e c i p i -r a t e d w i t h S r c k i n a s e . T h e f u n c t i o n a lc o n s e q u e n c e s o f s u c h i n t e r a c t i o n s h a v ey e t t o b e f u l l y d e f i n e d , b u t t h e y c o u l d ,f o r e x a m p l e , s e r v e t o p r o t e c t b e t a S r ca n d S r c - r e c r u i t e d R a f f r o m i n h i b k g r yp h o s p h o r y l a t i o n b y P KA 2S -2 L F u r t h e r -m o r e , P K A a c t i o n c a n h a v e p r o f o u n d e f -f e c t s o n c e l l a r c h i t e c t u r e a n d m o t i l i t y ,a n d t h u s , t h e l o c a l i s a t i o n o f a h i g h - a ff i ni t yc A M P - s p e c i f i c P D E t o c e l l c o r t i c a l r e -g i o n s m i g h t s u g g e s t a s p e c i f i c f u n c t i o n a lr o l e fo r s u c h i s o f o r m s .

T h e s e a r e j u s t tw o e x a m p l e s o f t h et a r g e t e d a s s o c i a t i o n o ! PD E 4 f a m i l y m e m -b e r s w i t h s u b c e i l u l a r s t r u c t u r e s . H o w -e v e r , o t h e r P D E 4 A i s o f o r m s s h o w d i s -t i n c t i n t r a c e ll u l a r l o c a l is a t i o n p a t t e r n s a sd o t h e l o n g f o r m s o f P D E 4 D . C o u p l e dw i t h t h e d i s t i n c t r e g u l a t o r y p r o p e r t i e sa n d c e l l u l a r e x p r e s s i o n p a t t e r n s , t h i s s u g-g e s t s t h a t P D E 4 i s o f o r m s m a y h a v e d i s -t i n c t f u n c t i o n a l r o l e s . B u t , t a r g e t e di n t r a c e l l u l a r l o c a l is a t i o n i s n o t a n e x c lu -s i v e p r o p e r t y o f t h e P DE 4 i s o f o r m s , a st h e t w o P D E3 i s o f o r m s a r e p r e d o m i n a n t l ym e m b r a n e - a s s o c i a t e d a n d a l t e r n a t i v em R N A s p l i c i n g o f P D E 2 g e n e r a t e s a( p l a s m a ) m e m b r a n e - a s s o c i a t e d is o f o rm5's.T h e i n t r a c e i l u la r l o c a l i s a t i o n o f p a r -t i c u l a r P D E i s o f o r m s i s l i k e l y t o p u t c o n -s t r a i n t s u p o n t h e i r e f f e c t i v e n e s s i n d e -c r e a s i n g i n t r a c e l l u l a r c A M P l e v e l s ( F ig . 3) .T h i s m i g h t o f fe r a n e x p l a n a t i o n a s t o

221

8/6/2019 Tailoring cAMP Signalling

6/8

EYlEWSC o m p a c tE x t r e m e ! r y p l o p h a n - r i c ha m i n o A m p h i p a t h i c M o b i l e h i n g e h y d r o p h o b i c A m p h i p a t h i ct e r m i n u s c o - he l ic a l d o m a i n d o m a i n d o m a i n h e l i c a l r e g i o n

" = - S e q u e n c eT a r g e t i n gr e g i o n

F igure 5The key fea tu res o f the am ino- te rmina l sp li ce reg ion o f the cAM P-spec i f i c phosph od i -es te rase P DE4A1, wh ich i s respons ib le fo r i n t race l l u la r ta rge t i ng o f th i s i so fo rm. The ' c o r eproduct , w i thou t any a l te rna t ive l y sp l i ced am ino- te rmina l reg ion , can be eng ineered andsubseq uent l y syn thes i zed as a h igh l y ac t ive , com p le te l y so lub le spec ies2z.4

w h y t h e s e l e c t i v e i n h i b i t i o n o f s p e c i f i cP D E i s o e n z y m e s i n c e l l s c a n a p p a r e n t l ylead to d is t in ct fun ct ion al ef fects ~8"3.Adenyla te yc lase I o ca l i u U o n , L i t t l e i sk n o w n a b o u t t h e l a te r a l d i s t r i b u t i o n o fa d e n y l a t e e y c l a s e i s o f o r m s w i t h i n t h ep l a n e o f t h e p l a s m a m e m b r a n e o f p o l a rce l l s 1. How ever , AC - Il l has bee n dem on -s t r a t e d ( F i g . 6 ) t o h a v e a p r o f o u n d l ya s y m m e t r i c d i s t ri b u t i o n i n c e r t a i n o l f ac -t o r y n e u r o n s , w h e r e i t i s e x c l u s i v e l y lo -c a i i s e d t o c i li a e x p o s e d a t t h e n a s a l c a v -i t y 1 . i t i s t empt i n g t o sug ges t t ha t on ei m p o r t a n t f e a t u r e o f d i v e r s i t y i n a d e n y l -a t e cyc l a s e l s o f o r m s i s t o a l l o w d i s t i n c tp a t t e r n s o f l a t e r a l s e g r e g a t i o n w i t h i nt h e p l a s m a m e m b r a n e . T h e m u l t i p l et r a n s c r i p t s e n c o d i n g b o t h A C -V a n dA C -V I m i g h t a l s o c o n t r i b u t e t o t h i s .Cemmta lkA n o t h e r r e a s o n fo r t h e d i v e r s i t y o fP D E a n d a d e n y l a t e c y c l a s e is o f o r m s a p -p e a r s t o b e t o p r o v i d e e n z y m e s w h o s ea c t i v i t y a n d l e v e l s c a n b e m o d u l a t e db y o t h e r s ig n a l t r a n s d u c t i o n s y s t e m s ,t h u s a l l o w i n g c e l ls t o ' t a i l o r ' th e i r c A M P -s i gna l l i ng sys t em ( T ab l e I ) .C alc iu m, P D E I e n z y m e s a r e e n c o d e d b yt h r e e g e n e s , w i t h a d d i t i o n a l i s o f o r m sg e n e r a t e d t h r o u g h a l t er n a t iv e m R N Aspl i c i ng3 2 . E a c h h a s t w o c a l m o d u l i n( C aM) b i nd i ng domai ns , whi ch a l l ow t he i ra c t i v i t y t o b e s t i m u l a t e d b y C a ~* . S u c hc r o s s t a l k h a s b e e n s h o w n t o b e i m p o r -t an t , f o r , : x a m p l e , i n r e g u l a t i n g c a r d i a c2 2 2

a d r e n e r g i c r e s p o n s e s w h e r e t h e i n -c r e a s e i n C a 2+ t r ig g e r e d t h r o u g h t h e~ a d r e n o c e p t o r a t t e n u a t e s t h e a b il i ty o ft h e ~ - a d r e n o c e p t o r - s t i m u l a t e d a d e n y l a t ec y c l a s e t o i n c r e a s e c A M P l e v e l s b yac t i va t i ng P DE 1.T he o pp or t u n i t y t o t une C a2~- regul at edP D E s y s t e m s , b y s e t t i n g d i f fe r e n t t h r e s h -o l ds f o r C a z ~ ac t i va t i o n , i s a l so o f f e r ed ,a s t h e v a r i o u s P D E I i s o f o r m s h a v e d i s -t i nc t s ens i t i v i t i e s t o a c t i va t i on by C a ~ "a n d t h i s c a n b e f u r t h e r m a n i p u l a t e d b ya l t e r n a ti v e s p l i c i n g o f P D E I C is o f o r m s as.A l t e r a ti o n s i n t h e s y n t h e s i s o f P DE is o -f o r m s a l s o a d d a n o t h e r d i m e n s i o n , i nt h a t w h i le P D E l C i s o f o r m s a p p e a r e q u a l l ye f f e c t i v e a t h y d r o l y s i n g c A M P a n dc G M P, t h e P D E 1 A a n d P D E1 B i s o f o r m sm a y s e r v e t o h y d r o l y s e c G M P p r e fe r e n-t ia ll y. T a i l o r ed cou p l i ng o f t he i n t e r ac t i ono f c A M P a n d c G M P s i g n a l li n g t o C a2+s i g n a l l i n g p a t h w a y s i s t h e r e f o r e p o s s -i b l e a t t he l eve l o f P DE .L i t t l e i s k n o w n a b o u t t h e c o n t r o l o fe x p r e s s io n o f t h e t h r e e P D E I g e n e s o rt he ce l l - spec i f i c gene r a t i on o f a l t e r na -t i v el y s p li c e d i s o i o r m s . H o w e v e r , r e c e n ts t u d i es o n C l iO c e l l s s4 h a w s h o w n t h a tP D E1 B c a n b e i n d u c e d w i t h t h e c h a r a c -t e r i st i c s o f a n ' i m m e d i a t e e a r l y ' g e n eu p o n r e c e p t o r - s t i m u l a t i o n o f li p id s i g -n a l l i n g s y s t e m s . T h i s p r o c e s s i s s e e m -i ng l y m e d i a t e d t h r o u g h t h e a c t i v a ti o n o fs p e c i f i c P K C i s o f o r m s , n a m e l y P K C - ~a n d P K C- e. T h u s , t h e i n d u c t i o n o f C a2+ -s t i m u l a t e d P D E 1 i s a c h i e v e d t h r o u g h

T I B S 2 2 - JUNE 199 7t h e a c t i o n o f r e c e p t o r s , w h i c h c a nt h e m s e l v e s i n c r e a s e i n t r a c e l l u l a r C a 2 ~.T h i s s i g n i f i e s a d e c i s i o n t o i m p o s e C a ~m o d u l a t i o n u p o n c y c l i c n u c l e o t i d e s i g -n a l l i n g i n t h e s e a c t i v a t e d c e l l s . T h eP D E1 fa m i l y a p p e a r s t h e n t o p r o v i d e ar o u t e f o r e l e v a t e d C a 2- l eve l s t o a t t enu-a t e b o t h cAMP - and cGM P - s i gna ll i ngp r o c e s s e s .B y c o n t r a s t t o t h i s , m u l t i p l e a d e n y l a t ec y c l a s e i s o f o r m s p r o v i d e t h e p o t e n t i a lf o r C a2 e i t h e r t o s y n e r g i s e o r a t t e n u a t ecAM P s i gna l l i ng 1,2. T h i s ac t i v i t y i s du e t oan i n t r i gu i ng d i ch o t o my , w i t h AC - I , -I11and : VIi i be i ng C a2+- s t i mui a t ed , whe r easAC-V an d -V! are CaZ -inhibi ted.L i tt le is k n o w n a b o u t t h e s i t e t h r o u g hw h i c h C a = ' + -C a M r e g u l a t e s a d e n y l a t ec y c l a s e a c t i v i t y . H o w e v e r , m u t a t i o n s i na p o t e n t i a l C a M - b i n d i n g d o m a i n i n t h eC i n t r ace l l u l a r l o op o f AC -I d i mi n i shC a2+- C aM sens i t i v i t y , cons i s t en t w i t ht h e i n v o l v e m e n t o f t h i s r e g i o n i n d e f in -i ng C a24- C aM r egu l a t i on . As f o r P DE 1,a l t e r na t i ve sp l i c i ng o f AC - Vl l l gene r a t e st h r e e d i s t i n c t i s o f o r m s t h a t v a r y i n t h e i rs e n s i t i v it y t o s t i m u l a t i o n b y C a2 + -C a M .C o n s i d e r a b l e e v i d e n c e s u p p o r t s t h ei d e a o f c o m p a r t m e n t a l i s e d C a 2 pools .T h i s m a y h a v e f u n c t i o n a l r e l e v a n c e t ot he r egu l a t i on o f AC - I, a s co - l oca l i s a t i ono f t h e e n z y m e w i th p l a s m a m e m b r a n eC a2 . c h a n n e l s h a s b e e n n o t e d as. S u c h as y s t e m a p p e a r s t o a l lo w C a z e n t r yt h r o u g h p l a sm a m e m b r a n e i on c h a n n e lst o s t i m u l a t e A C - I , w h e r e a s r e l e a s e o fC a z ' f r o m i n t ra c e U u l a r s t o r e s i n t o t h ec y t o s o | a p p e a r s t o b e i n e f f e c t i v e . T h i smay r e f l ec t an ac t i on o f i n i t i a l l y h i ghl oca l C az * c o n c e n t r a t i o n s o c c u r r i n g c l o s et o t h e p l a s m a m e m b r a n e , a s a r e s u l t o fc h a n n e l o p e n i n g t h e r e . C o n v e r s e ly , l o c a la c t i v a t i o n o f C a2 ' c h a n n e l s i n v e n t r i c u -l a r ce l l s , a s a r e su l t o f [ ~ - adr enocep t o ra c t io n , h a s b e e n s u g g e s t e d t o b e d e p e n d -e n t u p o n t h e c o m p a r t m e n t a l i s a t i o n o fi n t r a c e ll u l a r c A M Pa6.

C e r t a i n l y , s u c h c o m p a r t m e n t a l i s e dr e s p o n s e s w e r e a b o l i s h e d if P D E i n -h i b i t o r s w e r e p r e s e n t o r if t h e g e n e r i ca d e n y l a t e c y c l a s e - a c t i v a t o r f o r s k o l i nw a s u s e d i n s t e a d o f a [ 3 - a d r e n o c e p t o ra g o n i s t . T h i s i d e n t i f ie s a p i v o t a l r o l e n o to n l y f o r P D E s i n g e n e r a t i n g c o m p m ' t -m e n t a l i s e d c A M P r e s p o n s e s , b u t a l s of o r t h e a b i l i t y o f d i s t i n c t G - p r o t e i n -c o u p l e d r e c e p t o r s t o a c t i v a t e r e s t r i c te dp o o l s o f a d e n y l a t e c y c l a s e i n c e ll s , inc a r d i o c y t e s , th i s h a s b e e n n o t e d f o r t h ea c t io n o f ~ a d r e n o c e p t o r s a n d p r o s t a-g l a n d i n r e c e p t o r s . A l l o f w h i c h a d d s t om o u n t i n g e v i d e n c e f o r a k e y r o l e o fc A M P c o m p a r t m e n t a l i s a t io n i n ca r d i o-c y t e f u n c t i o n i n g ~6-3~.

8/6/2019 Tailoring cAMP Signalling

7/8

T I B S 2 2 - JUNE 1997

A n o t h e r e x c i t i n g f i n d i n g t h a t e m p h a -s i s e s s p a t i a l a s p e c t s o f b o t h c A M P a n dC aZ ~ -s ig n a~ l in g p ro cesses i s t h e r ecen to b s e r v a t i o n38 th a t , i n n eu r i t e s , cA M P cana l t e r t h e d i s t r i b u t i o n o l v e s i c l e s t h a t c a nr e l e a s e C a 2+ i n r e s p o n s e t o i n s u l i n a n d ,i n s o d o i n g , m o d u l a t e s e c r e t o r y p r o -c e s s e s a t t h e d i s t a l t i p o f t h e n e u r i t e s .I t w i l l b e i n t e r e s t i n g t o s e e h o w t h es y n t h e s i s p a t t e r n s o f t h e i r C a 2 + -s e n s it iv eP DE a n d A C e n z y m e s a r e c o - o r d i n a t e d i ns p e c i f i c c e i l t y p e s , a n d h o w t h e s e r e l a t et o t h e i n t e g r a t i o n o f C a2 + an d cA M P s ig -n a l l i n g . T h i s m i g h t b e o f p a r t i c u l a r i m -p o r t a n c e i n t h e c e n t r a l n e r v o u s s y s t e m ,w h e r e t h e s e e n z y m e s a r e p r o d u c e d a th i g h l e v e l s , a n d w h e r e c A M P a n d C a -~+a r e b e l i e v e d t o b e v e r y i m p o r t a n t i nl e a r n i n g p r o c e s s e s 39.

C y cl ic G ~ P. C y d i c n u c l e o t i d e P D E sp r o v i d e a p i v o t a l r o u t e f o r ' c r o s s t a l k 'r e g u l a t i o n o f c A M P s i g n a l l i n g b y c G M P .I n c r e a s e d s y n t h e s i s o f cG M P c a n b ea c h i e v e d t h r o u g h a t r i o p e p t i n b i n d i n g t os p e c if i c t r a n s m e m b r a n e r e c e p t o r s , w h i c ht h e m s e l v e s p o s s e s s g u a n y l a t e c y c l a s ea c t iv i ty , a n d a l s o t h r o u g h p r o c e s s e s t h a tg e n e r a t e N O , w h i c h c a n s t i m u l a t e s o l -u b l e g u a n y l a t e c y c l a s e . M e a n s f o r e i t h e rp o s i t i v e o r n e g a t i v e c o u p l i n g o f e l e v a t e dc G M P l e v e l s t o t h e c A M P - s i g n a ll i ng s y s -t e m c a n b e a c h i e v e d d e p e n d i n g u p o nt h e p a r t i c u l a r PD E i s of o r m s p r o d u c e d i nt h e t a r g e t c e l l s ,7 .8 . Thu s, PDE2 en zy m esh a v e a b i n d i n g s i t e f o r c G M P t h a t c o n -f e r s a p o s i t i v e h o m o t r o p i c s t i m u l a t o r yr e s p o n s e o n t h e c a t a l y ti c a c t i v a t i o n o ft h i s e n z y m e , d e c r e a s i n g i n t r a c e l l u l a rc A M P l e v e l s .H o w e v e r , a s P DE 2 c a n a l s o h y d r o l y s ec G M P, it s e f f e c t o n c y c l i c n u c l e o t i d e l e v -e l s i s l i k e l y t o b e r a t h e r c o m p l e x o w i n gt o c o m p e t i t i o n b e t w e e n c A M P a n d c G M Pa t t h e a c t i v e s i t e . B y c o n t r a s t , c G M Ps e r v e s a s a n e x t r e m e l y p o te n t c o m p e t i -t i v e i n h i b i t o r o f t h e c A M P - s p e c i f i c P D E 3e n z y m e , p r o v i d i n g a m e a n s f o r e l e v a t e dc G M P le v e l s t o p r o m o t e a n i n c r e a s e i nc A M P l e v e l s .Phospho ty la t ion .M o s t , i f n o t a l l , r e c e p -t o r s t h a t s t i m u l a t e a d e n y l a t e c y c l a s e a c -t i v i t y t h r o u g h G ~ b e c o m e r a p i d l y d e s e n -s i t i s e d s u b s e q u e n t t o t h e i r a c t i v a t i o n4'4t.T h e u n c o u p l i n g o f r e c e p t o r s f r o m G ~s t i m u l a t i o n o f t e n p r o v i d e s a k e y m e c h a -n i s m i n t h i s p r o c e s s . T h i s c a n b ea c h i e v e d b y r e c e p t o r p h o s p h o r y l a t i o na n d a l s o t h r o u g h i n t e r n a l i s a t i o n . H o w -e v e r , i n h i b i t i o n o f a d e n y i a t e c y c i a s e b yP K A - m e d i a t e d p h o s p h o r y l a t i o n , a s s e e nf o r AC -V , m i g h t a l s o p r o v i d e a k e y r o u t ei n s p e c i f ic c e l l t y p e s . N e v e r t h e l e s s , s i g -n a l t e r m i n a t i o n d e p e n d s i n h e r e n t l y o nl o w e r i n g i n t r a c e l l u l a r c A M P l e v e l s , a n d

REVIEWSP D Es p r o v i d e t i ae s o l e m e a n so f i n a c t i v a t i n g t h i s c y c l i c n u -d e o t i d e . T h ~ s , v p r e g u l a t i o no f PD E a c t i v i t y c a n b e e x -p e c t e d t o a i d in t h i s p r o c e s s .T h i s h a s b e e n d e m o n s t r a t e df o r P D E 3 a n d f o r c e r t a i nP D E 4 s p l i c e v a r i a n t s , i m p l y -i n g c e l l - ty p e s p e c i f i c u s e o fp r o m o t e r s t h a t d i f f e r e n t i a l l yr e g u l a t e P D E 4 i s o f o r m s y n -th e s i s s .a2. H o w ev er , t h e P K A -m e d i a t e d p h o s p h o r y l a t i o na n d a c t i v a t i o n o f b o t h P D E 3(Ref . 8 ) and PDE4D3 (Ref . 43 )c a n g i v e e x t r e m e l y r a p i d i n -c r e a s e s i n P D E a c t i v i ty . | n t h ec a s e o f P D E 3 , t h i s a c t i o n i sc r u c i a l i n t h e r e g u l a t i o n o fk e y p h y s i ol o g ic a l p r o c e s s e sin l i ve r , f a t an d h ea r t .A d e n y l a t e c y c l a s e a c t i v it yc a n b e r e g u l a t e d t h r o u g h t h eac t io n o f P K C . O r ig in a l ly , t h i sw a s i d e n t i f i e d t h r o u g h t h el o s s o f i n h i b i t o r y G~ c o u p l i n g ,w h i c h a p p e a r s t o b e a c h i e v e di n c e r t a i n c e l l s t h r o u g h t h ep h o s p h o r y l a t i o n o f ~ G ~ - 2 ,a n d i n o t h e r s b y a n a c t i o n o nt h e c a t a l y t i c u n i t o f a d e n y l -a t e c y c l a s e i t s e l f L 44. I n d e e d ,P K C c a n a c t i v a t e AC4/It/II[ i so -f o r m s , s e e n a s a n i n c r e a s e inth e V m ~ o f A C- II , w h i l e h av in gl i t t l e o r n o e f f ec t o n A C - IV /V /V I i s o f o r m s L T h e s i t e ( s ) o fp h o s p h o r y l a t i o n o n t h e s e A Ci s o f n rm s h a s y e t t o b e d e t e r -m i n e d , a s h a s a n y s p e c i f i c i t y f o r PK C is o -f o r m a c t i o n . T h u s , d e p e n d i n g o n w h i c hA C i s o f o r m s a r e p r o d u c e d , s t i m u l a t i o no f P KC c a n b e e x p e c t e d t o e x e r t v e r y d i f-f e r e n t e f f e c t s o n c A M P s i g n a l l i n g i n d i f-f e r e n t c e l l t y p e s . H o w e v e r , s u c h a c t i o n sa r e c o m p l i c a t e d b y i n h i b i t o r y a c t i o n s o fP KC on t h e c o u p l i n g o f c e r t a i n r e c e p t o r st o G s a n d G~, a s w e l l a s i n m o d u l a t i n g /i n d u c i n g P D E a c t i v i t y .A n i m p o r t a n t p h o s p h o r y l a t i o n e v e n ti n PD E r e g u l a t i o n r e l a t e s t o t h e a c t i v i t yo f P D E 3 a s i n d u c e d b y i n s u l i n , w h i c h i sp i v o t a l i n d e t e r m i n i n g t h e a n t i -l i p o ly t i ca c t i o n o f t h i s h o r m o n e s I n su l i n e l i c i t st h e p h o s p h o r y l a t i o n o f a s e r i n e r e s i d u eo n P D E 3 t h r o u g h a p r o c e s s t h a t c a n b ei n h i b i t e d b y w o r t m a n n i n , s u g g e s t i n g t h ei n v o l v e m e n t o f p h o s p h o i n o s i t i d e 3 - k i na s e .T h i s i s a l s o s i m i l a r t o t h e s t i m u l a t i c -, ofg l y c o g e n s y n t h a s e k i ~ ia s e b y i n s u l i n , a n di t i s t e m p t i n g t o s u g g e s t t h a t A E r t P K Bm i g h t b e i n v o l v e d i n PD E 3 r e g u l a t i o n .

P D E 3 h a s p r o v i d e d t h e f i r s t e x a m p l eo f m u l t i - s i t e p h o s p h o r y l a t i o n o f a P D E

n g u r e 6Dis t r i bu t i on o f aden y | a te cyc !ese i l l IAC- i I I) , phos phod i -e s t e r a s e 2 ( P D E 2 ) a n d P D E 4 A in o l f a c t o r y e p i t h e l i u m .P D E 4 A (g r e en ) a n d P D E 2 (r e d ) is o f o r m s a r e p r o d u c e din d i s t i n c t se ts o f neu rons . The o l fac to r y spe c i f i c AC-IHi s o f o r m i s r e s t r i c t e d t o t h e c i l ia , w h i c h l i n e t h e n a s a Jc a v i t y ( N C ) a n d w h i c h a s s o c i a t e w i t h t h e P D E 4 A -c o n t a i n i n g n e u ro n s . A x o n a l b u n d l e s ( A B ) e r e p o p u l a t e db y P D E 4 A a n d n o t P D E 2 . N o t s h o w n i s P D E 1 C 2 c o -I o c a l i s e d w i t h A C - I II , w h i c h i n d i c a t e s t h a t i n n e u r o n sc o n t a i n in g P D E 4 A a n d P D E I C 2 t h e s e i s o f o r m s a r e d if -fe ren t{a ll y Ioca l i sg d . The f igu re w as f r om J . Beavo anda d a p t e d f r o m R e f . 3 1 .

a s i t i s a l s o a c t i v a t e d a n d r e g u l a t e d n o to n l y b y P K A a n d a n i n s u l i n -r e g u l a t e dp r o t e i n k i n a s e , b u t a l s o b y a n a s y e tu n i d e n t i f i e d k i n a s e , w h i c h a p p e a r s t or e g u l a t e t h e P K A - m e d i a t e d a c t iv a t i o n o ft h i s e n z y m e 45.T h e m i t o g e n - a c t i v a t e d p r o t e i n ,,M A P)k i n a s e p a t h w a y m i g h t a l s o s e r v e a r o l ei n r e g v l a t i n g P D E a c t i v i t y a s , r e c e n t l y ,t h e P D E4 B2 i s o f o r m h a s b e e n s h o w n t ob e p h o s p h o r y l a t e d 46 a t a r e s i d u e w i t h i ni t s c a t a l y t i c r e g i o n b y M A P k i n a s e in v i tro ,a l t h o u g h t h e f u n c t i o n a l a n d p h y s i o l o g i -c a l s i g n i f i c a n c e o f t h i s i s n o t y e t c l e a r .f i - p r o t e l n { ~ , / r e g u l a t i o n . A l l a d e n y l a t e c y -c l a s e i s o e n z y m P s a l e a c t i v a t e d b y G T P -b o u n d c og a n d i n h i b i t e d b y a d e n o s i n ea n a l o g u e s t h r o u g h t h e ' P - s i t e 1 . H ow -e v e r , t h e y a r e d i f f e r e n t i a ll y r e g u l a t e d b yC ~ p r ot e in ~ / - s u b u n i t s . T h u s , f o r e x a m p l e ,1 3 " / s u b u n it s i n h i b i t A C -I w h i l e s y n e r g i s -i n g w i t h t h e a c t i v a t i o n o f A C - Ii a n d A C -I Vp r o m o t e d b y G sc LT h i s l a t t e r o b s e r v a t i o ne x p l a i n s w h y , u n d e r c e r t a i n c o n d i t i o n s ,t h e a c t i v a t i o n o f A C - I I / I V b y G s c a n b e

223

8/6/2019 Tailoring cAMP Signalling

8/8

V I E W Snnpl i f i ed by the action of other G pro-Ie ins ( in c lud ing the G,- like subfam i ly ) ,which are genera l ly cons idered as inh ib i -Iors of adenyla te cyc!a se activity, throughIhe release of th eir G-f3,/subunits. Thesignif icance of this com plex state of affairshas not been reso lved , as has any spec i -ficity regarding t he five forms of [3- and atleast ten form s of "y G-protein su bun its .Insight in to t he s i t e o f ~ , , in terac t ionon AC-II has c om e a bout f rom s tud iesshowing that a pept ide represent ingresidu es 956-9 82 in the intrace!lu!ar seg-m ent C., of AC-II can disrupt [3~/ regu-lat ion o [ not on ly AC-II, but als o of o the r13-/ -modulated sys tem s , inc lud ing pho s -p h o l i p a s e C - [ 33 a n d t h e mu s c a r i n i cace ty lcho l ine regu la t ion o f the GIRK Kchann e ls in th e heart I :. It would see mthat he terotr imer ic G prote ins can rap-id ly re lease [3~ / subun i t s , w hich canthen be spa t ia l ly Ioca l i sed and can sh owboth d ivers i ty and se lec t iv i ty in the irin terac t ion , prov id ing a sophis t i ca tedregu la tory sys tem .ConclusionMultiple forms of both cAMP-PDEsand adeny]ate cyclase allow cells totai lor their cAMP-signall ing system andto a l low spec i f i c dynamic adjus tments .The in trace llu lar loca l i sa t ion o f the s ig -na l generators , t erminators and de tec -tors i s de termined so as to a f fec t com-partmenta l i sed responses wi thin a s ing lecell type. This confers the ability tobreach the PKA activation thresho l ( i bye i ther or both the activation of adenyl-ate cyc las e or inh ib i t ion o f specific PDEl so forms . The ident i f i ca t ion o f a ll the secomponents , toge ther wi th the de f in i -t ion of their spatial Iocalisation and regu-la tory propert ie s shoukl a l low us toapprec ia te the p le thora o f d i s t inc t pro-cesse s that cAM P s igna l l ing contro l s . In

particular, it should allow us to definehow cAMPs igna l l ing is organ ised wi th inthe three -d im ens iona l matr ix o f spec i f i cce l l types . Th is i s e s sent ia l i f we are tounders tand aberrat ions that occur ind isease s ta tes , and i f we are to exp lo i tth i s sys tem e f fec t ive ly in deve lop ingnove l therapeut ic agents .AcknowledgementsWork in the auth ors ' laborator ies i ssupported by grants f rom the Medica lRe se a rc h Co u n c i l ( U K ) a n d t h eWeHcome Trust . We thank 1. Ramsdenfor he lp in the generat ion o f the f iguresin this article.References

1 Sunahara. R. K. . Dessauer . C. W. an dGi lman. A. G. 11996) Annu. R oy. PharmacoLToxicol. 3 6 . 4 6 1 - 4 8 02 Call . J. J.. Parekh. R. S. and K rup insk i, J . ( 1996)J. Bio l . Chem. 2 7 1 . 1 0 8 9 - 1 0 9 53 Zhang. G. . Liu. Y. . Ruoho. A. E. and Hur ley , J. H.~1997) Nature 3 8 6 . 2 4 7 ~ 2 5 34 Taussig. R. and Gi lman. A. G. (1995) J. Biol.Chem. 2 7 0 . 1 - 45 Beavo. J. A. (1995) Physio l . Rev. 7 5 . 7 2 5 - 7 4 86 8o l ger . G . ( 1994 ) Cell. S~gnal. 6 . 8 5 1 - 8 5 97 Cont i . M. . Nemoz. G. . Set te. C. and Vicini , E.L1995) Endocr. Roy. 1 6 . 3 7 0 - 3 8 98 Manganiel lo. V. C. et al. { 1 9 9 5 } Arch. Biochem.Biopl]ys. 3 2 2 , 1 - 1 39 Pieroni. J. P. e ta ] . 11995) J. Biol. Chem. 2 7 0 .2 1 3 6 8 - 2 1 3 7 320 A dic. E. J. and M i i l igan. G. ( /1994) Biochem. J.3 0 3 , 8 0 3 - 8 0 8I /1 MacEwan. D. J. . K im. G D. and Mi l l igan. G( 1 9 9 5 ) MoL Pt~armacol. 4 8 . 3 1 6 - 3 2 512 Heyw orth. C. M. . Wal lace A. V. an dI ' ]oustay. M D. (19 83 ) B i o ch e m J . 2 1 4 . 9 9 1 1 023 Houslay. M D {1990b Cel l . S igna l 2 . 8 5 b 824 Faux. M. C. and Scot t . J. D. (199 6) C e l l 8 5 . 9 1 225 Rubin. C S. L19941 Btochim . Bnoph.Fs. 4ctai 2 2 4 , 4 6 7 - 4 7 916 Hempel . C . M. et al. ! 1 9 9 6 ) Nature384. 1 6 6 - 1 6 927 K lauck . T . M . and Scot t . J . D . ( 1995 ) Cell.Signal. 7 . 7 4 7 ~7 5 71 8 Bacska~. B. J. et al. ( 1 9 9 3 ) Science 2 6 0 . 2 2 2 - 2 2 619 Smi th . K . J. et al. { 1 9 9 6 i g . Biol . Chem. 2 7 1 .1 6 7 0 3 - 1 6 7 1 12 0 Bolger . G. . McPhee. I . and Houslay. M. D.

TIBS 22 - JUNE 1997

( 1 9 9 6 ) J Bio l . Chem. 2 7 1 . 1 0 6 5 - 1 0 7 121 McPhee. I . et at. ( 1 9 9 5 ) Biochem. J. 3 1 0 .9 6 5 - 9 7 42 2 Pooley. L. . Shakur . Y. . Rena. G. an dHous l ay. M. D . ( 1997 ) Biochem. J. 2 7 1 . 1 7 7 - 1 8 52 3 Huston. E. et a l . ( 1 9 9 6 ) J . Biol . Chem. 2 7 1 .3 1 3 3 4 - 3 1 3 4 42 4 O'ConnelL C. et al. ( 1 9 9 6 ) Biochem. J. 3 1 8 .2 5 5 - 2 6 22 5 Hafner. S . , Ad l er . H. S . and M i schak . H. ( 199 4)MoL Cell. Biol. 1 4 . 6 6 9 6 - 7 0 32 6 Cook. S . and McCormi ck. R (199 3) Science2 6 2 . 1 0 6 9 - 1 0 7 22 7 Parsons. J. and Weber , M. J . ( 1989) Curt. Top.Microbio l . Immunol. 1 4 7 . 8 0 - 1 2 72 8 Mul ler . T . . Engeis. R and Fozard. J. R. (1996)

Trends PharmacoL Sci. 1 7 . 2 9 4 - 2 9 82 9 Erdogan. S . and Hous l ay. M. D . ( 19 97)Biechem. J. 321. 1 6 5 - 1 7 53 0 Seldon. P. M. . Barnes. R J. . Me]a. K. an dGiembycz , M. A . 11995) Mol. Pharmacol. 4 8 .7 4 7 - 7 5 731 Jui l fs. D. ora l . ( 1 9 9 7 ) Proc. Natl . Acaa. Sct.U . S. A . 9 4 . 3 3 8 8 - 3 3 9 53 2 Rybalkin. S. D. and Beavo. J. A. (1996)B~ochem. Soc. Trans. 2 4 . 1 0 0 5 - 1 0 0 93 3 Yah. C. . Zhao. A. Z. Bent ley. J. K. and Beavo . J. A.( 1 9 9 6 ) J. Biol. Chem. 2 7 1 . 2 5 6 9 9 - 2 5 7 0 634 Spence. S . et al. ( 1 9 9 7 ) Biocl~em. J. 3 2 1 .1 5 7 - 1 6 33 5 Fagan. K. A. . Mahey. R. and Cooper , D. M. F.( 1 9 9 6 ) J . Biol . Chem. 2 7 1 . 1 2 4 3 3 - 1 2 4 4 43 6 Jurevic ius. J. and Fischmeister , R. (19 96 ) Proc.Natl . Acad. S ci . U. S . A . 9 3 . 2 9 5 - 2 9 93 7 Aass. H. . Skom edaL T. and Osnes. J. B. (19881J. MoL Cel l . Cardio l . 2 0 . 8 4 7 - 8 6 03 8 Jonas. E. A. et a l . i 1 9 9 7 ) Nature 3 8 5 . 3 4 3 - 3 4 639 Cooper . D. M. F. . Mons. N. and Karpen , J. W.( 1 9 9 5 ) Nature 3 7 4 . 4 2 1 - 4 2 44 0 L o h s e . M J . ! 1 9 9 3 l Bioct~m~. Bioph!.s Acta1 1 7 9 . 1 7 1 - 1 8 841 Benovic. J. L. et aL ( 1 9 8 9 ) Science 2 4 6 . 2 3 5 - 2 4 04 2 Torphy. T. J. et al. ( 1 9 9 5 ) J . Biol . Chem. 2 7 0 ,2 3 5 9 8 - 2 3 6 0 44 3 Sot t c , C , and Conh, M, (1996) J , Blot, Cr~em.2 7 1 . / 1 6 5 2 6 - 1 6 5 3 4. l . l Morr i t , . N J. . 8u~,hf ic ld. M. and Houslay. M. D.( 1 9 9 6 ) Biocl~em.J . 3 1 5 . 4 1 7 ~4 2 045 Ki lgour . E. . Anderson. N. G. and Houslay. M. D.{ 1 9 8 9 ) Biochem. J. 2 6 0 . 2 7 3 64 6 Lenhard. J. M. et a l . ( 1 9 9 6 ) Biochem. J, 3 1 6 .7 5 1 - 7 5 84 7 Chert . J. et at. ( 1 9 9 5 ) Science 2 6 8 . 1 1 6 6 - 1 1 6 94 8 Hous l ay . M. D . ( 1985) Proc. Nutr. Sac . 4 4 ,1 5 7 - 5 6 54 9 Shakur . Y. et at. ( 1 9 9 5 ) Biochem. J. 3 0 6 .8 0 1 - 8 0 9

A sM o f ~c isco , C A , US A(" r th u r K o m b e r g ,w r ,~ 2 1 s t C e n t u r y ' )s c o p e o f t h e|U fo r t h e W B M B .

A p o s t e r b y P e e r B o r k , J M g S c h u l ~ a n d C h r i s P o r rt |n g , il lu s t r a t in g t h e

H o c h e m i s t r y a n dla tio n a l Co n g ress

2 2 4