TIBS 12 - February 1987 63

The molecular basis of odor recognition

Doron Lancet and Llmberto Pace

Following dec n,,l~ of sauty and spectdamm, the molecula, mechamsms of olfactton are begin. tung to be tmdersfood Odorant receptors appear to actwate a cyclic nucleolute enzyme cascade, including a G TP.bmdmg protein, analogous wuh the processes of hormone, neuro-

mmmutter and vmed reception

A typical olfactory epithelium contmns 107 structurally similar, bipolar sensory neurons with long clha emanating from their dendmes (see Anholt's accom- panying amcle, Fig 1) Olfactory ctha are the site of sensory transductton, akin to visual rod and cone outer segments Different sensory cells gwe the same response to different odorants - depolarization and the finng of action potentials 1 2. Thus, it was suggested that different olfactory neurons express dis- tinct olfactory receptor (OR) molecules, which activate a common transducUon chain (Fig 1)

Olfactory transdectien: cyclic AMP cascade

One of the rewarding recent develop- ments tn olfactory biochemistry has been the eluadatmn of an olfactory transduc- tlon mechanism In analogy with neuro- transmitter and hormone slgnahng in neurons and other cells 3 4, and with hght activation of photoreceptor cells 5 (Fig 2), accumulating data suggest that cyclic AMP (cAMP) serves as second mes- senger in odorant activation of the chemosensory neurons

Electrophyslologlcal studies show that cAMP (or its membrane penetrable analogs), phosphodtesterase inhtbltors, and guanine nucleotldes can ehclt odor- ant-hke responses or modulate odorant activation t 2,6 7 Biochemical studies, carried out in cdmry-ennched membrane preparations (Refs 8 and 9 and refer- ences cited thereto) analogous to iso- lated retinal rod outer segments, confirm and extend these results The isolated chemosensory organelles of frog 9-n and rat tl-i3 contain a very high specific activ- ity of adenylate cyclase, and the clhary enzyme is actwated 1 5-2 5-fold by odor- ants at physiological concentrationst°-~ This odorant response is tissue- and hgand-specafic and GTP-dependent, as

D Lancet and U Pace are at the Department o f Membrane Research, The Wet27nann Insumte o f Scteuce, Rehovot, Israel

expected for a second messenger- generating enzyme coupled to a specific receptor wa a GTP-bmd, ng protein (G protein)3. 4

Odorant rmxtures are more efficient than mdwldual r, oorants in activating adenylate cycla~¢ ~0, consistent with the notmn of several OR molecule classes converging or~ a common transductlon mechanism Additional corroboration of receptor heterogeneity comes from the low value of Hill's coefficient (~0 4) dis- played by the odorant activation dose- response curves t°

Adenylate cyclase measurements m isolated olfactory ctha prowde the first cell-free assay for olfactory actwa- tton 214 Sklar e~ a/If were able to con- duct a rapid screening and comparison of

the activation properhes of many dozens of odorants usm- ,he assay (see 'Other transduction mechanisms') This assay also showed that gp95, a major trans- membrane glycoprotem of olfactory ciha s 15 16, might be involved in odorant reception (see 'Olfactory receptor isola- lion') An obwous advantage of the adenylate cyclase assay over ligand bind- ing techniques is that only functionally Important interactions are registered Non-specific binding, which presumably does not lead to transductlon events, remains undetected

Ogactory G protein The GTP dependence of odorant acti-

vation suggests that guanine nucleotlde- binding protein (G protein) is involved in the couphng between OR and adenylate cyclase Olfactory G protein appears to be similar to G~, the stlmulatory G pro- rein of some hormone and neuro- transmitter receptors ~, since odorants enhance rather than dmunish cAMP production Also, cholera toxin- catalysed [32p]ADP nhosylatmn of olfac- tory cdsa of frog 1017 and rat I-' labels a polypeptlde substrate of 42-45 kDa resembling the o-subumt of Gs, and leads to actwatmn of adenylate cyclase in the clhary membranes r- The same G,, polypeptlde can be seen through interac-

sensory cell types S1,S2 S1,S3,S4 S5 •-. S2,S N

o,,oc,o receptor proteins -

second ; messenger GTP other generation GDP -," Jp enzymes ,', /

P

mem brahe transduction

Fig i A schemntw view o f olfactory receptwn ,4t least a fe,~ dozen ( M) olf~ctor~ receptor molecule IORJ types may exmst, present on the dendrites o f a similar number IN) o f ~ensor~ neuron t~ pes (see Refi 2 26 and 37) A sensor, neuron ( eg SI~ may have more thml one t~pe o f receptor molecule (m this case OR I and OR ~ A given OR molecule ( eg OR,_) ma, be present on more tha, r one semorv neuronal tvpe ( m this case S t, St and S.O Altemauvely, clonal exclnslon may pertain, where a one-to-one relauoashrp adl hold fe g sensory neuron 5~ and OR molecule OR # Many OR molecule types ma~ com erge (m dtfferem cells) on a common trarL,ducuon machinery, the best candrdate being G~ and adenvlate c; close (see re:Ill c 4 MP for other messengers produced b) parallel transduruon en."vmes ) tt ould then actt; ate the sensorz trot channels

to produce neuronal membrane depolanzauon ~ ) 19~7 El,,c,,zer Science Pubhsht.r~ B ~ AmslO'dam i)lITh - ~1l~7r8 "1 ~12 Igl

64 T I B S 12 - February 1987

=ion with G prote[]-specttic antisera t4,=7 In flog, olfactory Gm, hke adenylate cyc- lase ts enriched [] olfactory ctha com- pared to membranes from hver or from decthated eptthehum t° 12 t7 Other G protein types, the intubttory (G~) and the brmn-speafic (Go) are present I° 12, but are not parucularly enriched m olfactory ctha These prote[]s could be related to modulation of the odorant response, possible by endogenously secreted hgands is

Gs has been shown to eyast [] at least two molecular forms, with a-subumts of 42-45 kDa and 48-52 kDa ~. produced by alternative mRNA sphcmg 19 Olfactory csha con=am mainly an ADP-nbosylated a-subantt sandar, but possibly not identi- cal to, the lower molecular weight form 12 Olfactory G s appears to differ from G, m brmn or hver [] having higher guan[]e nucleottde affimty 1° 12 and dif- ferent relative potency of actlvauon by stereolsomenc gua[]ne nucleonde analogs 14 Such differences could be due either to gene-related ammo acid sequence vanauons or to post-transla- tional modtficatmn

Polynucleoudes coding for G protems [] a rat olfactory epithelial eDNA hbrary have been sequenced, and clones corres- ponding to G s, Gt and G o, as well as at least two other G proteins, identified 2U At present tt ~s not clear wluch of these sequences corresponds to the functmnal component of the sensory neurons

Interest[]g support for the proposed role of G s [] olfactmn comes from studies of the human geneuc deficiency dis- ease, pseudohypoparathyroichsm (PI-IP) PHP type Ia patients, who are reststam to the action of several cAMP-mediated hormones, and have only about 50% of the normal G~ level, are tound to have an imp•red olfactory capacity 21 It is suggested that the sensory impairment =s due to a defect [] the peripheral recep- tmn mecha[]sm lfso, these data may be taken to imply that olfactory G Sts either =denucal with hormonal G s, or that both are under common genetic control

Other transduction mechanisms Is adenylate cyclase activation the

only mecha[]sm of olfactory transduc- =ion 9 Many odorants (notably those [] the fruity, floral, mmty and herbaceous classes) activate adenylate cyclase, but other physiologically active odorants (e g putrid ahphatm .,ctds, ammes and orgamc solvents) do not affect the enzyme in ,solated clha tt Thus, certa[] OR molecules may generate an lntra- cellular signal through different trans- ductmn mechanisms Guanylate cyclase and cyclic nucleotide phosphodlesterase

in rat olfactory eptthehal membranes were found to be odorant []sensitive t3 Another possible mechamsm ss modula- tion of phosphattdyl •osttol metabol- ism 22, supported by ewdence whereby L-alamne, an anuno acid odorant, acti- vates phosphatidyl mosRol-4,5-b=sphos- phate phosphodmsterase (phosphohpase C) in isolated fish olfactory clha 23

There are other possible transductlon mecha[]sms (1) The direct modulation by some odorants of ton channels m the sensory neuronal membranes 24, stmdar to the mechamsm of action, of the •cot=[]c acetylchohne receptor (see Anholt's article for a further discussion) Such a mechamsm lacks the amphfica- uon provided by second messenger enzyme cascades, but may be s=g[]fi- candy faster* (2) Receptor-mdependent odorant modulation of the transduction machinery, e g direct activation of olfactory G protem and/or adenylate cyclase proteins by tluol reagents Is The latter could account for the potent and rather umform odor of most suL~ydryl- contmnmg odorants

Olfactory receptor diversity An mterest[]g proble[] =s how so

many odorants with widely diverse []olecular structures are agomsts, t e are capable of []ducmg the correct con- format,onal transmons necessary for receptor activatmn in other receptor systems, only a few []embers of any given group of structurally related hgands can serve as agomsts In contrast, practmally any chemmal modification of a gwen odorant will yield another odorant, though often with a different perceived quality Two possible explana- tions are. (1) OR molecules have par- tmulafly broad selectmty ranges, (2) there ts a rather large number of differ- ent OR protein types Because of the stnlong resemblance between olfactory transduction mechamsms and those of other G protem-acttvatmg receptors, it ~s less probable that OR molecules are unique [] thew allostenc properties It appears more hkely that OR molecules constitute a repertoire of many different receptor types, each with the usual narrow ago•s t range

It has been postulated that OR protems constitute products of a multigene fam- ily, similar to that of =mmunoglobullns (see Ref 2 for a review) Members of this famdy, would be expressed [] differ- ent sensory cells, either in a 'one cell - one receptor' type arrangement or

*The speed of the olfactory response (100-1(300 ms) is consistent with both a d.¢ct mechanism and a second messenger cascade 2

otherwise, accountmg for the dwerstty of odorant responses of []dmdual sensory cells Like tmmunoglobuhns and T-cell receptors 25, OR molecules could have variable regions contaimng the odorant bmdmg rues and share a constant regmn responmble for transductton (Fig 1)

What is the possible raze of olfactory r ecep to r r epe r to i re 9 Data on human specific anosnua~ (genetic defects [] the abthty to perceive certmn odorants) mdl- cate the existence of at least a few dozen receptor types 26 The variations of elec- trophysmlogmally momtored s[]gle-cell responses in the olfactory eplthehum are consment with this nummum number=,2 Based on hgand affimty cons,deratmns tt has been argued 2as that the upper Imut for the olfactory repertoire may be 102-104 , considerably smaller than the tmmunoglobulm riper=one (107-109, Ref 25) lntngumgly, color vmon oper- ates with only three photoreceptor type s27, possibly because absorption spectra of orgamc chromophores can easily cover one-third of the entire v~s- ible wavelength range, wlule a typical protein receptor would usually b[]d only a much smaller fraction of all possthle hgandst

Olfactory receptor isolation The neuronal membrane receptors

that transduce odorant signals have not yet been unequivocally identified The difficulties [] acluevmg tins goal by hgand-blnd[]g techmques may be attn- buted to the possible dwerstty of OR blnd[]g sRes, as well as to the expected high levels of non-specific blnchng due to odorant hpophthclty and presumed weak affi[]ty 2. A pyrazme-b[]d[]g pro- tern isolated from olfactory eplthehal tis- sue was found to be water-soluble and present m relabvely Ingh concentrations in secretory glands and mucus of nasal epltheha (Ref 29 and references cited therein) It probably acts as an odorant career or scavenger facdttat[]g access to or removal from the receptors An olfac- tory eptthehal camphor b=ndmg activity 3° remams to be identified as a defined polypeptide species

An alternative approach to the iden- tification of OR proteins ts to screen the sensory ctha membranes for polypeptide candidates with receptor properties other than odorant blnchng Useful cntena can be those features which are common to other G protem-acttvat=ng receptors trans[]embrane disposition,

=Once a photon ,s absorbed, photo~somenzed reunal may mduce a similar conformaUonal change to that caused by odorant bmdlng Notably. many potent odorants are ,soprenoid compounds, similar to retmal ~

TIBS 12 - February 1987

glycosylatron , tissue specticq and mteracnon wth G, and adenylate cyclase A promismg receptor can&date IS glycoprotem gp95, the only specific polypeptlde of frog olfactory cdla that fulfills many of these cTytena2,s 15 16 This glycoprotein IS a major diary compo- nent, whose bdayer concentration agrees Hnth c@lunts of freeze fracture intramem- branous partlcles suggested to corres- pond to OR moleculessl Electro- physlolog& and cell-free adenylate cyc- lase studies employmg specific lectms and antIbodIes provide support for Its mteractum wth the olfactory transduc- tion machmeryl4 Homologs of gp95 appear to be present m other vertebrate speaesl6. and its future molecular gene- tic charactenzatlon may provide Impor- tant clues on olfactory receptio&Is Specifically, it wdl be important to deter- mme whether gp95 (or any other recep- tor can&date) displays heterogeneity at the protein and gene levels

FramcAMPtoionchuds The major mtraceuular target of the

second messenger CAMP IS CAMP- dependent protein kmase. ‘IIus enzyme IS present m olfactory &a and m declh- ated epithelral membranes32 It is ach- vated by nucromolar concentrabons of CAMP, and by GTPyS, which presum- ably acts through endogenous CAMP generation (cGMP IS a much less potent activator ) Several clhary polypephde substrates, notably two with molecular masses of 24 and 26 kDa (pp24 and pp26), have been ldentlfied through their CAMP-dependent phosphoryla- tlonJ2 These results stimulate further studies of the mvolvement of CAMP- dependent protein phosphorylatlon m olfactory transduchon m parallel to its role m other neuromodulatlon pro- cesses33

The other can&date transducmg enzyme, phosphohpase C, hberates two second messengers, mosltol tnsphos- phate and Bacylglycerol, the latter actlvatmg protein hnase C (Ref 22) Such kmase actlvlty was Investigated In olfactory membranes, with confhctmg results Anholt et al I7 Idenhfied protem kmase C a frog olfactory cdla by lmmunoblot analysis and by phorbol ester bmdmg However, neither they, nor Heldman and Lancet”, could demonstrate protein kmase C atinty by phosphorylatlon assays m the presence of calcium and phosphatldylsenne

Elect,ophysdo@cal and blochenucal data support the notion that changes of mtracellular CAMP modulate membrane Ion conductance Yet, httle IS known about the nature of the Ion channel(s)

Involved, or about the mechamsm of theu modulation by CAMP 0ao1 shmu- latlon leads to depolanzatron and increased membrane conductance In olfactory neurons’, suggestmg the open- ing of catlon channels The best candl- date for the transduction current tamer IS sodium. but potassium has also been Implicated (see accompanymg review by Anholt)

In addition to actmg through phos- phorylation by CAMP-dependent pro- tem kmase, CAMP could act through duect allostenc modulation of Ion chan- nels, in analogy wth the function of the cGMP-gated sodium channels of retmal rod outer segment (reviewed m Ref 8; Fig 2) Evidence for the latter mechamsm has recently been obtained through smgle ion channel recordmgsU

Photon

Rh 3

GMP cGMP4

Odoront

65

Olfactory adaptation Electrophynolo~cal signals recorded

from the penpheral sensory neurons of olfactory eplthebum exhlblt partial adaptation wthm 1 s from the onset of stimulus Tlus process appears to be dls- tact from the well-known disappearance of odor sensation upon longer (- 1 mm) exposure, which IS thought to be mediated by central nervous system pro- cessing Fast olfactory adaptation may be akm to desensltlzatlon processes In other G protem-linked receptors In other systems, the photoreceptor protein rhodopsm and P-adrenerguz receptor, molecular detads of such adaptation processes have been elucrdated (renewed III Refs 5 and 35) Smcc OR molecules appear to share functional propertles with both P-AR and rhodopsm (Ref 2.

B-Agonist

I

9ARK

ATP cAMPt

?g t No /Ca

w Gs AC

ATP c+MPt

Fig 2 A hypothettcal view o/rheposstble homology between o@cforv r~eplor (OR) molecules and orher cychc nucleonde-coupled receptors. bdrenergrc receptar @AR) and the phororemptor protem rhodopsrn (Rh) Odomnt molecules serve an analogous role to ~gonrsts or to photons, acavanng a ret umdenn)ied OR protein OR protems may belong to the group of membrane receptors wth seven transmembmne domarnr that mcludes BAR and rhodopsm IS Ltke pA R, OR pr otemacnvatea G,-adenylate qclase svstem

rncreasrng theproducnon of CAMP/mm ATP Analogoudy. photolysed rhodopsvl acts ares a transducm-

phosphodrestemsesystem to mcrease the breakdown of cGMP Two dtstmct mechanisms mediate the modu-

lanon of wn channel conductance bv cychc nucleotrdes In some neurons, CA MPmav cause the opening or

ton channels bv acnvatrng CAMP-dependent prorem kuaase whtch catalyses prottw phosphonlonons’ In

rod outer segments cGMP activates a canon conductance bv dwectly mtemcnng with an mn channel’ One

or both of these mechanwm may underhe theodomnt-related acttvatton of a mnon conduaancem dfactov

neurons OR protetns could undergo phosphorylanon bv an oljacton receptor-spenfic kmaw” (labelled ORK). hypothesrzed to be homologousto PAR kmase @ARK) and to rhodopsm krnare (RhK)

66 T I B S 12 - February 1987

Fig 2), It would be mterest lng to look for phosphoryla t lon of olfactory cilia polypepttdes by receptor-specific kln- ases tha t act only on act ivated recep- tors 35 In parallel, c A M P - d e p e n d e n t pro tem lanase and protein kmase C could phosphorylate t ransduct lon com- ponen t s and play a role in chemosensory adapta t ion (see Ref 35)

Conclusion Future research of olfactory recept ion

will probably culminate in the Identi- fication, ~solation and study of O R molecules A most rewardmg result of such accomphshment could be a com- plete mapping of O R genes m the h u m a n genome, helping to elucidate the molecular genetic basis of specific ol- factory deficiencies and popula t ion polymorphlsms, slmdar to the recent achievements m the field of color photo- reception 27 Because there may be 10- 100 t imes more genes Involved, the degree of indlwdual v a n a t m n In O R genes may be extreme, possibly compar- able to that found in lustocompaUblhty genes which have been shown to be func- tionally related to odor recept ion 36

Studies of O R genes can greatly benefit from research on Drosophi la me lanogas - ter specl f ic anosmla m u t a n t s 37 Extending the biochemical and geneuc studies of O R molecules to insects may provide a crucial source of knowledge for the study of the mechanisms invoh,ed In insect p h e r o m o n e detection 3s The example set by studies of olfactory receptmn may pave the way to a molecular eluc~datlon of other chemoreceptaon systems no- tably taste and vomeronasal reception, both deahng vath sttmuh del ivered m aquo , the latter possibly const i tut ing a 'pept~derglc' olfactory-hke system 39

Unravel ing O R genes may lead to a be t te r unders tanding of gene expression m developing or regenerat ing olfactory sensory neurons This should help resolve open questions such as how olfactory neurons form the correct synap t¢ contacts depending on the i r O R specificity 2 Thus olfactory neurons may become a useful model for studies of neuronal connectivity and function as well as for molecular t ransduct lon mechamsms

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