Identification of a Transferable Two-Amino-AcidMotif (GT) Present in the C-Terminal Tail of theHuman Lutropin Receptor that RedirectsInternalized G Protein-Coupled Receptors froma Degradation to a Recycling Pathway

COLETTE GALET, LE MIN, RAMESH NARAYANAN, MIKIKO KISHI, NANCY L. WEIGEL, AND

MARIO ASCOLI

Department of Pharmacology (C.G., L.M., M.K., M.A.), The University of Iowa, Iowa City, Iowa52242-1109; and Department of Molecular and Cellular Biology (R.N., N.L.W.), Baylor Collegeof Medicine, Houston, Texas 77030

Although highly homologous in amino acid se-quence, the agonist-receptor complexes formedby the human lutropin receptor (hLHR) and rat (r)LHR follow different intracellular routes. Theagonist-rLHR complex is routed mostly to a lyso-somal degradation pathway whereas a substantialportion of the agonist-hLHR complex is routed to arecycling pathway. In a previous study, we showedthat grafting a five-residue sequence (GTALL)present in the C-terminal tail of the hLHR into theequivalent position of the rLHR redirects a sub-stantial portion of the internalized agonist-rLHRcomplex to a recycling pathway.

Using a number of mutations of the GTALL motif,we now show that only the first two residues (GT)of this motif are necessary and sufficient to inducerecycling of the internalized agonist-rLHR com-

plex. Phosphoamino acid analysis and mutationsof the GT motif show that phosphorylation of thethreonine residue is not necessary for recycling.Lastly, we show that addition of portions of theC-terminal tail of the hLHR that include the GTmotif to the C-terminal tails of the rat follitropin ormurine �-opioid receptors promotes the post-endocytotic recycling of these G protein-coupledreceptors.

We conclude that the GT motif present in theC-terminal tail of the hLHR is a transferable motifthat promotes the postendocytotic recycling ofseveral G protein-coupled receptors and that theGT-induced recycling does not require the phos-phorylation of the threonine residue. (MolecularEndocrinology 17: 411–422, 2003)

INTERNALIZATION OF G PROTEIN-coupled recep-tors (GPCRs) is one of the many consequences of

agonist-induced GPCR activation. Although much hasbeen learned about the pathways by which GPCRs areinternalized (reviewed in Refs. 1–4), much less is knownabout the pathways that determine the fate of theinternalized receptors (reviewed in Refs. 3–5). Mostinternalized GPCRs are quickly recycled back to theplasma membrane (3–5), but a few such as the rodentor porcine lutropin receptors (LHRs) (6–10), the humanthrombin receptor (11, 12), the murine �-opioid recep-tor (mDOR) (13, 14), and the human endothelin B re-ceptor (15–17) are instead directed toward a lysoso-mal degradation pathway.

The rat LHR (rLHR) and human LHR (hLHR) providea rather unique tool to understand the structural fea-

tures of GPCRs that determine their intracellular fatesbecause they share a high degree of amino acid se-quence homology (18), yet they follow a divergent fateonce internalized (10). The internalized agonist-hLHRcomplex is routed mostly to a recycling pathway,whereas the internalized agonist-rLHR complex isrouted mostly to a degradation pathway (10). Usingchimeras and exchange mutants of these two recep-tors, we showed previously that, when grafted into theC-terminal tail of the rLHR, a five-amino-acid residuemotif (GTALL) present in the C-terminal tail of thehLHR can redirect the internalized agonist-rLHR com-plex from a degradation to a recycling pathway (10).The GTALL motif is interesting because it shares twostructural features (a phosphate acceptor and adileucine sequence) with the C-terminal tetrapeptide(DSLL) of the �2-adrenergic receptor (�2-AR), a motifthat is known to be important for the recycling of theinternalized �2-AR (19, 20). Like the GTALL motif,the DSLL motif is also transferable in that it can reroutethe internalized rLHR (10) and the internalized mDOR(14) from a degradation to a recycling pathway. Be-cause serine/threonine phosphorylation (19, 21) and

Abbreviations: �2-AR, �2-Adrenergic receptor; EBP, ezrinbinding protein; FSHR, FSH receptor; GPCRs, G protein-coupled receptors; HA, influenza hemagglutinin epitope;hCG, human chorionic gonadotropin; hLHR, human LHR;LHR, lutropin receptor; mDOR, murine �-opioid receptors;NHERF, sodium-hydrogen exchange regulatory factor; rLHR,rat LHR; rFSHR, rat FSHR; wt, wild-type.

0888-8809/03/$15.00/0 Molecular Endocrinology 17(3):411–422Printed in U.S.A. Copyright © 2003 by The Endocrine Society

doi: 10.1210/me.2002-0161

411

dileucine motifs (19, 20) have been implicated as im-portant determinants of the fate of internalized recep-tors, the experiments described here were designed tobetter define the structural features of the GTALL motifthat induce the recycling of the internalized agonist-rLHR complex.

RESULTS

The GT Motif from the GTALL Sequence of thehLHR C-Terminal Tail Is Necessary and Sufficientto Reroute the Human Chorionic Gonadotropin(hCG)/rLHR Complex from a Degradation to aRecycling Pathway

To better define the structural features of the GTALLmotif that are involved in recycling, several new mu-tants were constructed (Fig. 1, Table 1) and theirintracellular routing determined in transiently trans-fected cells that had been allowed to internalize 125I-hCG (10). Because the hCG-LHR complex does notdissociate after internalization (6, 7, 9), the fate of thereceptor can be conveniently and accurately quanti-tated by following the fate of the radioactive ligand(6, 9, 10).

The results summarized in Table 1 show that, asreported previously (10), substitution of the QPIPPsequence of the C-terminal tail of the rLHR with thecorresponding GTALL sequence of the C-terminal tailof the hLHR reroutes a substantial portion of the in-ternalized hCG-rLHR complex from a degradation to arecycling pathway. More importantly, the data pre-sented in Table 1 also show that grafting only thedileucine motif of the GTALL sequence (i.e. rLHR-LL)does not reroute the internalized hCG-rLHR complexto a recycling pathway, whereas grafting the phos-phate-acceptor motif (GTA) of the GTALL sequence

(i.e. rLHR-GTA) does induce recycling. Several addi-tional mutants of the GTA motif were then analyzed todetermine which of these three amino acids were nec-essary to induce the recycling of the rLHR. The resultspresented in Table 1 clearly indicate that only the firsttwo amino acids (GT) of this motif are necessary andsufficient to reroute the hCG-rLHR complex from adegradation to a recycling pathway.

As shown in Table 1, the routing of the hLHR orrLHR to a recycling or degradation pathways is notcomplete. Whereas the majority of the rLHR is de-graded, the internalized hLHR is distributed to thedegradation and recycling pathways in roughly equalproportions. We believe that this is due to the exper-imental conditions used. Because sorting to a recy-cling pathway is not 100% efficient and because therecycled hCG remains bound to the receptor (see Ta-ble 1 and Refs. 6, 7, and 10), the recycled hormoneundergoes additional rounds of internalization leadingto the eventual degradation of substantial amounts ofthe internalized hormone. Importantly, however, theextent of recycling and degradation of 125I-hCG isbasically the same in cells expressing the hLHR-wildtype (wt) or rLHR-GT (Table 1).

The fate of the internalized rLHR-wt, hLHR-wt, andrLHR-GT were also directly ascertained using confocalmicroscopy in cells cotransfected with the myc-taggedreceptors and Rab5a-GFP (an endosomal marker, seeRef. 22) or procathepsin D-GFP (a lysosomal marker, seeRef. 23). The results presented in Fig. 2 show that in cellsincubated without hCG the rLHR-wt and rLHR-GT arelocalized mostly to intracellular compartments, whereasthe hLHR-wt is localized mostly at the plasma mem-brane. The large intracellular pools of rLHR-wt andrLHR-GT do not colocalize with either Rab5a-GFP orprocathepsin D-GFP and are likely to represent the im-mature 68-kDa LHR precursor, which is localized in theendoplasmic reticulum (reviewed in Ref. 18). This inter-

Fig. 1. Amino Acid Sequence Alignment of the C-Terminal Tails of the hLHR, rLHR, rFSHR, and mDORThe sequences for the hLHR (accession no. P22888), rLHR (accession no. P16235), rFSHR (accession no. AAA41175), and

mDOR (accession no. NP038650) were obtained from the National Center for Biotechnology Information data bank and werealigned using ClustalW. Only partial sequences are shown starting at the NPXXY motif present in transmembrane helix 7 (TM-7)that is highly conserved among GPCRs of the rhodopsin/�2-AR subfamily of GPCRs (56). The partial box at the left end of thesequences shows the cytoplasmic end of TM-7. A cysteine present in the C-terminal tail that is highly conserved among GPCRsof the rhodopsin/�2-AR subfamily is also outlined by a box. Residues that are identical in at least two of the four sequences shownare highlighted in gray. Dashes indicate gaps introduced for optimum alignment. The serine residues that become phosphorylatedupon agonist stimulation of the rLHR and hLHR are marked with asterisks (29, 30, 33). The box outlined with double lines highlightsthe GTALL motif of the hLHR and the corresponding sequence of the rLHR. The mDOR/hLHR chimera was produced by replacingthe residues marked with the arrows at the bottom of the mDOR sequence with the hLHR residues by the arrow at the top of thehLHR sequence.

412 Mol Endocrinol, March 2003, 17(3):411–422 Galet et al. • Sorting of the Internalized LHR

pretation is in agreement with results obtained by West-ern blots which show that the intracellular (i.e. 68 kDa)precursor of the LHR is much more prevalent in cellstransfected with the rLHR-wt and rLHR-GT than in cellstransfected with the hLHR (see Fig. 3 and Refs. 18 and24). Although the presence of the large intracellular poolof rLHR precursor poses a problem in visualizing thesurface rLHR that becomes internalized after hCG stim-ulation, colocalization experiments with the endosomaland lysosomal markers clearly show that such internal-ization does takes place (Fig. 2). After addition of hCG, aportion of the rLHR-wt redistributes into intracellularcompartments that are rich in Rab5a (i.e. endosomes) aswell as intracellular compartments that are rich in pro-cathepsin D (i.e. lysosomes). In contrast, after addition ofhCG, the rLHR-GT redistributes into endosomes but notinto lysosomes (Fig. 2). The data presented in Fig. 2 alsoshow that the internalization of the cell surface hLHR-wtis easier to visualize because the relative abundance ofthe intracellular hLHR precursor is low compared withthat of the mature cell surface hLHR. After addition ofhCG, the internalized hLHR-wt redistributes into endo-somes but not into lysosomes.

These data are in complete agreement with the dataon the fate of the internalized 125I-hCG summarized inTable 1 and show under that hCG stimulation theinternalized rLHR-wt is sorted to endosomes and ly-sosomes, whereas the internalized hLHR-wt andrLHR-GT are sorted only to endosomes.

The Addition of the GT Motif Prevents the Down-Regulation of the rLHR

An agonist-induced decrease in the cell surface LHR isanother assay that can be used to discern the fate ofthe LHR after agonist stimulation. Because the extentof agonist-induced decrease of the cell surface LHR(down-regulation) is dictated by the rate of internaliza-tion of the receptor vs. the rate of recycling of theinternalized receptor (6, 25, 26), we predicted that thehCG-induced down-regulation of the cell surfacerLHR-GT mutant would be less than that of therLHR-wt if the rates of internalization of hCG mediatedby the rLHR-wt and by rLHR-GT are comparable.

To test this prediction, we used an immunoprecipi-tation/immunoblotting approach to measure the levelsof the cell surface LHR after stimulation with hCG.Because the immunoprecipitation is done using wholecell lysates, three species of the LHR can be detected(reviewed in Ref. 18). The 68-kDa band is much moreprevalent in cells expressing the rLHR (see left andmiddle panels of Fig. 3) than in cells expressing thehLHR (see right panel of Fig. 3) and it represents anintracellular, immature precursor of the LHR that isthought to be localized in the endoplasmic reticulum(also see Fig. 2 above). The 85-kDa band is much moreprevalent in cells expressing the hLHR (see right panelof Fig. 3) than in cells expressing the rLHR (see left andmiddle panels of Fig. 3), and it represents the mature

Table 1. Fate of the Internalized 125I-hCG-LHR Complex in 293T Transiently Transfected with the rLHR-wt, hLHR-wt, orMutants Thereof

ReceptorRelevant

Amino AcidSequence

125I-hCG (% of Cell-Associated Radioactivity at t � 0)

Cell Associated Released

Intracellular Surface Bound Degraded Undegraded

rLHR-wt QPIPP 31 � 1 12 � 1 47 � 2 10 � 1hLHR-wt GTALL 34 � 1 30 � 1 28 � 1 8 � 1rLHR/hLHR Exchange Mutantsa

rLHR-GTALL GTALL 31 � 4 29 � 1 35 � 5 5 � 1rLHR-LL QPILL 29 � 8 12 � 1 55 � 8 5 � 1rLHR-GTA GTAPP 32 � 1 32 � 1 29 � 1 8 � 1rLHR-QTA QTAPP 28 � 1 11 � 1 51 � 2 10 � 2rLHR-GT GTIPP 31 � 2 29 � 1 33 � 3 7 � 1rLHR-QT QTIPP 32 � 2 12 � 1 47 � 3 9 � 1rLHR-GP GPIPP 28 � 1 12 � 2 51 � 1 9 � 1Additional Mutations of rLHR-GTAb

rLHR-GAA GAAPP 29 � 5 14 � 1 45 � 3 7 � 1rLHR-GSA GSAPP 29 � 2 10 � 1 51 � 1 9 � 2rLHR-GEA GEAPP 28 � 3 15 � 1 47 � 2 10 � 2rLHR-GDA GDAPP 32 � 1 2 � 1 57 � 1 8 � 1

Cells were transiently transfected with the indicated constructs. The fate of the internalized 125I-hCG-LHR complexes wasmeasured as described in Materials and Methods. Results are expressed as a percent of the total intracellular 125I-hCGradioactivity present at t � 0 (10,000–20,000 cpm/well in individual experiments), and they represent the mean � SEM of 3–14independent transfections.a The sequence alignment illustrated in Fig. 1 show that the QPIPP sequence near the C terminus of the rLHR corresponds to theGTALL sequence of the hLHR. All constructs shown are mutants of the rLHR in which the QPIPP sequence (or portions thereof)was substituted for the corresponding sequence present in the hLHR. The residues substituted are shown in bold.b The threonine residues of the GTA motif of rLHR-GTA was mutated as indicated to the residues shown in italics.

Galet et al. • Sorting of the Internalized LHR Mol Endocrinol, March 2003, 17(3):411–422 413

cell surface LHR. The 165-kDa LHR is mostly an ag-gregate of the 68-kDa immature receptor, which isagain more prevalent in cells expressing the rLHR thanin cells expressing the hLHR.

As expected, incubation of the cells with hCG resultedin a decrease in the density of the 85-kDa band (i.e. themature cell surface LHR) but had no effect on the inten-sity of the intracellular LHR precursors. The intensity of

Fig. 2. Colocalization of the rLHR-wt, rLHR-GT, and hLHR-wt with Rab5a and Cathepsin D293T cells were cotransfected with the indicated myc-LHR construct and Rab5a-GFP (left panels) or the indicated myc-LHR

construct and procathepsin D-GFP (right panels). The transfected cells were washed and incubated with (52 nM) or without hCGat 37 C for 2 h as indicated. The cells were fixed and the receptors (in red) were visualized using an anti-myc monoclonal antibody(9E10) and a CY5-conjugated antimouse antibody. Rab5a-GFP and procathepsin D-GFP are shown in green, and colocalizedcomponents are shown in yellow. The cells were observed and analyzed using a Bio-Rad Laboratories, Inc. confocal microscopeat the Central Microscopy Facility of The University of Iowa.

414 Mol Endocrinol, March 2003, 17(3):411–422 Galet et al. • Sorting of the Internalized LHR

the 85-kDa band was thus measured in several experi-ments before and after agonist stimulation, and a sum-mary of the quantitative data obtained is presented inTable 2. This table also displays the rates of internaliza-tion of hCG mediated by each of the three receptors.These results show that, in spite of a slightly faster rate ofinternalization, a property that enhances down-regula-tion (26), the extent of hCG-induced down-regulation ofthe cell surface rLHR-GT is much lower than that of thecell surface rLHR-wt as predicted by the divergent fatesof the internalized receptors. Also, as shown previously(27), the half-time of internalization of the hLHR-wt ismuch shorter than that of the rLHR-wt.

When considered together, the results presented inTables 1 and 2 and Figs. 2 and 3 clearly show that thefates of the hormone and the receptor are different forrLHR-wt and rLHR-GT and that the properties ofrLHR-GT are very similar to that of the hLHR-wt. Most ofthe hCG internalized by rLHR-wt is degraded, the recep-tor can be localized to lysosomes, and there is a sub-stantial decrease in the density of the cell surface rLHR-wt. In contrast, a substantial portion of the hCGinternalized by the rLHR-GT or hLHR-wt is recycled,these receptors do not localize to lysosomes, and thereis a minimal decrease in the amount of cell surface LHR.

The Rerouting of the rLHR to a RecyclingPathway by the Addition of the GT SequenceDoes Not Require Threonine Phosphorylation

Because the hCG-induced activation of the rLHR andthe hLHR results in the phosphorylation of severalsites present in their C-terminal tails (28–30), and theGT sequence contains a phosphate acceptor (threo-nine), we considered the possibility that the phosphor-

ylation of the threonine present in the GT sequence isnecessary for recycling. This hypothesis was tested byperforming phosphoamino acid analysis on rLHR-wt,hLHR-wt, and the rLHR-GT mutant as well as by mu-tagenesis of the GT sequence.

Because previous phosphoamino acid analysis andmutagenesis studies of the rLHR have shown that thisreceptor is phosphorylated mostly on serine residues(28, 29, 31), we reasoned that if the threonine present inthe GT motif is phosphorylated we should observe anincrease in the phosphothreonine content of therLHR-GT when compared with the rLRH-wt. The resultssummarized in Fig. 4 show that this is not the case. Theamount of phosphothreonine detectable in the rLHR-wtis minimal, and there is no increase in the phosphothreo-nine content of the rLHR-GT mutant when comparedwith rLHR-wt. In addition, and in agreement with previ-ous mutagenesis studies (30) the amount of phospho-threonine detected in the hLHR-wt, a recycling receptorthat contains the GT sequence as part of its wild-typeC-terminal tail is also minimal (Fig. 4).

As an independent and complementary test for thepossible involvement of threonine phosphorylation onthe recycling of the rLHR-GT mutant, we assessed thebehavior of an additional mutant in which the threonineof the GT motif was mutated to another phosphateacceptor (S) that can be phosphorylated by serine/threonine protein kinases (32). Table 1 shows that aGS motif did not support the recycling of the rLHR. Inaddition, substitution of the threonine residue of theGT motif by residues that would mimic the charge ofphosphoserine or phosphothreonine (aspartate or glu-tamate) failed to support the recycling of the rLHR

Fig. 3. Agonist-Induced Down-Regulation of the Cell Sur-face LHR

Transiently transfected 293T cells were stimulated with asaturating concentration of hCG (52 nM) and lysed immedi-ately or after a 6 h incubation at 37 C. Lysates were immu-noprecipitated with the 9E10 antibody and the amount ofimmunoprecipitated receptor was visualized on Westernblots using the 9E10 antibody covalently coupled to horse-radish peroxidase as described in Materials and Methods.

Table 2. Rates of Internalization of hCG and hCG-InducedDown-Regulation of the Cell Surface LHR in 293T CellsExpressing the rLHR-wt and rLHR-GT

Receptor t1/2 of Internalization(min)a

hCG-InducedDown-Regulation ofthe Cell Surface LHR

(% of the control)b

myc-rLHR-wt 135 � 25 21 � 12myc-rLHR-GT 111 � 8 83 � 19myc-hLHR-wt 21 � 6 83 � 9

a The rate of internalization of hCG was measured as describedin Materials and Methods. The results shown represent themean � SEM of three to four independent transfections.b Transiently transfected cells were stimulated with hCG (52 nM)and lysed immediately or after a 6-h incubation at 37 C. Lysateswere immunoprecipitated with the 9E10 antibody and theamount of immunoprecipitated receptor was visualized andquantitated by Western blots as described in Materials andMethods and shown in Fig. 3. To correct for loading and vari-ability in receptor expression, the amount of cell surface recep-tor (i.e. 85 kDa) was normalized to the amount of receptorprecursor (i.e. 68 kDa) and the normalized data from the cellsincubated with hCG for 6 h were expressed as % of the cor-rected data from the cells that had hCG added but processedimmediately (see Fig. 3). The results shown represent themean � SEM of six independent transfections.

Galet et al. • Sorting of the Internalized LHR Mol Endocrinol, March 2003, 17(3):411–422 415

(Table 1). Substitution of the threonine residue of theGT motif by alanine also did not support recycling ofthe rLHR (Table 1).

Lastly, previously characterized mutants of the rLHRor the hLHR that were rendered phosphorylation-deficient by simultaneous mutation or removal ofseveral C-terminal tail serine residues that becomephosphorylated upon agonist activation (shown in redin Fig. 1, also see Refs. 10, 29, 30, and 33) were shownto follow the same fate as their wild-type counterparts(data not shown and Ref. 10).

Taken together, these data allow us to conclude thatthe phosphorylation of the threonine residue of the GTmotif, or the phosphorylation of other serine residuespresent in the C-terminal tail of the rLHR or hLHR, arenot involved in determining the fate of the internalizedreceptors.

The Recycling Properties of the GT Motif AreTransferable to Other GPCRs

Because the sorting of internalized GPCRs is dictatedmostly by their C-terminal tails (10, 12, 14, 16, 20,34–36), one would predict that the portion of the C-

terminal tail of the hLHR that contains the GT motifshould be able to redirect another internalized GPCRfrom a degradation to a recycling pathway. This pos-sibility was tested by examining the properties of mu-tants of the mDOR and the rat FSH receptor (rFSHR)modified to express the GT sequence at their C-terminal tails.

The mDOR is another GPCR that is routed to alysosomal degradation pathway upon agonist-induced internalization (13, 14). This GPCR was cho-sen as a model to further ascertain the recycling prop-erties of the GT motif because it is only distantlyrelated to the LHR (see alignment in Fig. 1) and be-cause recent studies have shown that replacing its last6 residues with the last 10 residues of the �2-ARredirect the internalized mDOR from a degradation toa recycling pathway (14). Thus, we prepared a similarconstruct (designated mDOR/hLHR) in which the last 6residues of the C-terminal tail of the mDOR were re-placed with the last 17 residues of the C-terminal tail ofthe hLHR. The fates of HA-tagged versions of themDOR-wt and the mDOR/hLHR constructs transientlyexpressed in 293T cells were compared by confocalimaging. The confocal micrographs shown in Fig. 5show that, as expected (13, 14), most of the influenzahemagglutinin epitope (HA)-mDOR-wt is localized atthe surface of transiently transfected 293T and thatagonist stimulation results in a redistribution of themDOR from the cell surface to Rab5a-rich compart-ments (i.e. endosomes) and procathepsin D-rich com-partments (i.e. lysosomes). The HA-mDOR/hLHR isalso localized mostly at the cell surface, but agoniststimulation results in a redistribution of this receptorfrom the cell surface to endosomes and not to lyso-somes. These data clearly show that a portion of theC-terminal tail of the hLHR that encompasses the GTmotif promotes the postendocytotic recycling of an-other GPCR (the mDOR) that is normally routed to alysosomal degradation pathway.

The LHR and the FSHR are members of the sameGPCR subfamily (18), and although their amino acidsequence identity at the C-terminal tail is only approx-imately 40% (see Fig. 1), the internalized rFSHR isrouted mostly to a recycling pathway. Thus, an addi-tional test for the recycling properties of the GT motifwas conducted by examining the effects of the C-terminal tail of the rLHR with or without the GT motif onthe routing of the internalized 125I-hFSH/rFSHR com-plex. The results of these experiments are presented inTable 3 and show that most of the 125I-hFSH internal-ized by the rFSHR is recycled back to the medium inintact form. These data also show that grafting theC-terminal tail of the rLHR into the rFSHR (i.e. the FFLchimera) reroutes some of the internalized 125I-hFSHto a degradation pathway. Because the rate of hFSHdegradation is slower than the rate of recycling, thererouting effect of the C-terminal tail of the rLHR de-tected in the FFL chimera is not only reflected by adecrease in the amount of undegraded hormone andan increase in the amount of degraded hormone re-

Fig. 4. Phosphoaminoacid Analysis of the rLHR-wt, hLHR-wt, and rLHR-GT

Phosphoaminoacid analysis of the indicated receptors wasperformed on receptors immunoprecipitated from agonist-stim-ulated cells that had been prelabeled with 32Pi as described inMaterials and Methods. The position of migration of authenticstandards is also shown. Only the region of the thin layer platescontaining the phosphoamino acids is shown.

416 Mol Endocrinol, March 2003, 17(3):411–422 Galet et al. • Sorting of the Internalized LHR

leased into the medium, but also by an increase in theamount of intracellular hormone retained by the cells.More importantly, however, the data presented in Ta-ble 3 also show that the rerouting effect of the C-terminal tail of the rLHR does not occur when the QPsequence is mutated to a GT sequence (i.e. theFFL-GT chimera). Thus, the C-terminal tail of the rLHRmodified to contain a GT sequence promotes the re-cycling of the rLHR and also the rFSHR.

DISCUSSION

Once internalized, the agonist-activated GPCRs canrecycle back to the plasma membrane or accumulatein lysosomes where they are degraded (reviewed inRefs. 3–5). Several studies have shown that the pos-tendocytotic sorting of GPCRs varies greatly between

receptors and that it is determined mainly by structuralfeatures present within their C-terminal tails (10, 12,14, 16, 20, 34–36). More recently, experiments con-ducted with the �2-AR and the LHR have identified twodistinct but homologous motifs, DSLL, the C-terminaltetrapeptide of the �2-AR, and GTALL, a motif near theC terminus of the hLHR that can mediate the recyclingof internalized GPCRs (10, 14, 19, 20).

Although all investigators agree that the DSLL motifis essential for the recycling of the internalized �2-AR(19, 20) and that it can induce the recycling of nonre-cycling GPCRs when added to their C-terminal tails(10, 14), there is no agreement on the identity of thecellular protein(s) that mediate the DSLL-dependentGPCR recycling. Cao et al. (19) implicated a PDZ do-main protein known as ezrin binding protein (EBP)50or sodium-hydrogen exchange regulatory factor asbeing responsible for this event, whereas we (10) and

Fig. 5. Colocalization of the mDOR-wt and mDOR/hLHR with Rab5a and Cathepsin D293T cells were cotransfected with the indicated HA-mDOR construct and Rab5a-GFP (left panels) or the indicated myc-LHR

construct and procathepsin D-GFP (right panels). The transfected cells were washed and incubated with (10 �M) or withoutDADLE ([D-Ala2, D-Leu5 enkephalin], an mDOR agonist) at 37 C for 30 min as indicated. The cells were fixed and the receptors(in red) were visualized using an anti-HA monoclonal antibody (12CA5) and a CY5-conjugated antimouse antibody. Rab5a-GFPand cathepsin D-GFP are shown in green and colocalized components are shown in yellow. The cells were observed and analyzedusing a Bio-Rad Laboratories, Inc. confocal microscope at the Central Microscopy Facility of The University of Iowa.

Galet et al. • Sorting of the Internalized LHR Mol Endocrinol, March 2003, 17(3):411–422 417

Cong et al. (20) have presented evidence that excludeEBP50/NHERF from being involved in this process.Lastly, Cong et al. (20) have presented evidence thatimplicates a protein that participates in membranefusion (N-ethylmaleimide-sensitive factor) instead ofEBP50/NHERF as being responsible for the DSLL-dependent recycling of the internalized �2-AR. A gen-eral role for the DSLL motif in the recycling of inter-nalized GPCRs is unlikely, however, because mostinternalized GPCRs are routed to a recycling pathway(3–5), but there is only one other GPCR (the P2Y1purinergic receptor, see Ref. 37) that has a similarC-terminal sequence (D-S/T-x-L). Based on theseconsiderations, it appears likely that there may beother motifs present in the C-terminal tails of GPCRsthat are involved in determining the fate of the inter-nalized receptors.

Structure function studies on the fates of the inter-nalized hLHR and rLHR can provide important dataabout this issue because, in spite of a high degree ofamino acid sequence identity, the agonist-activatedhLHR and rLHR follow a different fate once internal-ized. The internalized hCG-rLHR complex is routedmostly to a lysosomal degradation pathway, whereas

the internalized hCG-hLHR complex is routed mostlyto a recycling pathway (this paper and Ref. 10). Wehave previously concluded (10) that the lysosomalrouting of the internalized rLHR is due to the lack ofsorting motif(s) needed for recycling rather than thepresence of sorting motif(s) needed for degradation forthree reasons. First, progressive deletions of the C-terminal tail of the rLHR fail to reroute the internalizedhCG-rLHR complex from a degradation to a recyclingpathway (9). Second, grafting the C-terminal tail of therLHR into the hLHR does not reroute the internalizedhCG-hLHR complex from a recycling to a degradationpathway (10). Third, addition of the DSLL C-terminaltetrapeptide of the �2-AR to the extreme C-terminus ofthe rLHR, grafting the C-terminal tail of the hLHR intothe rLHR, or grafting short sequences, GTALL or GT,present in the C-terminal tail of the hLHR into thecorresponding position of the C-terminal tail of therLHR reroutes the internalized agonist-rLHR complexfrom a degradation pathway to a recycling pathway(this paper and Ref. 10).

Because of the obvious structural similarities be-tween the GTALL and the DSLL motifs (they both havea phosphate acceptor and a leucine dimer), it wasreasonable to hypothesize that one or both of thesefeatures may play a ubiquitous role in determining thefate of internalized GPCRs. The data presented hereshow that the leucine dimer of the GTALL motif is notneeded for recycling and identify the GT sequence asbeing necessary and sufficient to induce the recyclingof the internalized agonist-rLHR complex. Our resultsalso show that the threonine residue present in the GTmotif does not need to be phosphorylated for recy-cling to occur. We show that there is no increase inthreonine phosphorylation of the rLHR-GT when com-pared with the rLHR-wt and that there is little or nothreonine phosphorylation in the hLHR. Moreover,substitution of the threonine of the GT motif with an-other phosphate acceptor (serine) or with two acidicamino acids that mimic the negative charge of phos-phothreonine or phosphoserine do not support recy-cling. These results stand in contrast with theDSLL-dependent recycling of the �2-AR or the DSLL-induced recycling of the rLHR both of which are dis-rupted by mutations of the leucine dimer (10, 20). Inaddition, the phosphate acceptor (serine) of the DSLLmotif of the �2-AR can be phosphorylated by GRK5 invitro (38) and overexpression of GRK5 reportedly in-hibits the recycling of the internalized �2-AR (19).Moreover, because mutation of the serine of the DSLLmotif to a residue that cannot be phosphorylated (ala-nine) or to one that mimics the charge of the phos-phorylated serine (aspartic acid) disrupted recycling ofthe internalized �2-AR and the in vitro associationof the �2-AR with EBP50/NHERF it was proposed thatthe GRK-5 catalyzed phosphorylation of the serine ofthe DSLL motif disrupted recycling by preventing theassociation of the �2-AR with EBP50/NHERF (19). Theeffects of a serine to threonine exchange were notexamined, however. When considered together, the

Table 3. Fate of the Internalized 125I-hFSH in 293TTransiently Transfected with the rFSHR-wt, the FLL and theFFL(GT) Chimeras

Receptor

125I-hFSH(% of Cell-Associated Radioactivity at t � 0)

Cell Associated Released

Intracellular SurfaceBound Degraded Undegraded

rFSHR-wt 26 � 1 5 � 1 14 � 2 55 � 3FFLa 41 � 2 2 � 1 24 � 3 34 � 2FFL-GT 27 � 2 3 � 1 13 � 1 56 � 1

a The binding, signaling, and internalization properties of theFFL chimera have been reported elsewhere (43).Cells were transiently transfected with the indicated con-structs. The fate of the internalized 125I-hFSH complexes wasmeasured as described for 125I-hCG in Materials and Meth-ods. The only exception was that an excess of eFSH (500ng/ml) was included in the medium during the second incu-bation to prevent rebinding of any undegraded 125I-hFSHreleased into the medium. Results are expressed as a percentof the total intracellular 125I-hFSH radioactivity present att � 0 (10,000–20,000 cpm/well in individual experiments),and they represent the mean � SEM of three independenttransfections. The fate of the 125I-hFSH internalized by therFSHR is basically the same as that of the 125I-hCG internal-ized by the hLHR. The only reason why recycling of the125I-hFSH is seen as the release of intact 125I-hFSH into themedium instead of an increase in the amount of cell surfacehormone (as shown in Table 2 for hCG) is because the ex-periments involving FSH were done in the presence of anexcess of FSH in the medium during the second incubation(to prevent the rebinding of any released 125I-hFSH, seeabove), whereas those with hCG did not include an excess ofhCG in the medium during the second incubation (see Ma-terials and Methods).

418 Mol Endocrinol, March 2003, 17(3):411–422 Galet et al. • Sorting of the Internalized LHR

data on the differential effects of mutations of theleucine dimer and the phosphate acceptor imply thatthe mechanisms by which the GT and the DSLL motifsinduce GPCR recycling are rather different.

Another important difference between the DSLL andGT motifs is the role that they play in their nativecontext (i.e. the �2-AR and the hLHR, respectively).Whereas mutation or removal of the DSLL motif dis-rupt the recycling of the internalized �2-AR (see aboveand Refs. 19 and 20), mutation or removal of the GTmotif of the hLHR does not reroute the internalizedagonist-hLHR complex from a recycling to a degrada-tion pathway (10). As already discussed elsewhere(10), the apparent lack of involvement of the GT motifin the recycling of the internalized hLHR may be ex-plained by the presence of redundant sorting motifs inthe hLHR. Because the C-terminal tail of the rLHRdoes not appear to have any sorting motifs that pro-mote recycling or degradation (as discussed above),then the grafting of recycling motifs, such as the GT orthe DSLL motifs, on the C-terminal tail of the rLHRwould be sufficient to reroute most of the internalizedrLHR to a recycling pathway.

An important similarity of the DSLL and GT motifs isthat their recycling properties are transferable to otherGPCRs. As shown here for the GT motif and elsewherefor the DSLL motif (10, 14), both of these motifs inducethe recycling of the rLHR and the mDOR, two distantlyrelated GPCRs that are normally sorted to a lysosomaldegradation pathway. Moreover, we also show herethat grafting the C-terminal tail of the rLHR into an-other recycling GPCR (the rFSHR) reroutes portion ofthe internalized rFSHR to a degradation pathway un-less a GT motif is included in the C-terminal tail of therLHR. When the C-terminal tail of the rLHR grafted intothe rFSHR is modified to contain a GT motif, however,the recycling properties of the rFSHR are preserved.

As already outlined above, a general role for theDSLL motif in GPCR recycling is unlikely becausemost internalized GPCRs recycle but do not terminatewith a DSLL or homologous sequence, and the GTmotif identified here is too short to be used as a queryof GPCR databases to determine if its presence cor-relates with GPCR recycling. Fortuitously, however,we noticed that this motif is present in the C-terminaltail of the type A endothelin receptor and it is absent inthe type B endothelin receptor and the presence ofthis motif correlates with the fate of the internalizedendothelin receptors (see Fig. 1 of Ref. 16). Thus, theagonist-activated endothelin B receptor is targetedto a lysosomal degradation pathway, whereas theagonist-activated endothelin A receptor is targeted toa recycling pathway (15–17, 39). Interestingly, switch-ing the C-terminal tails of these two receptors wasalso shown to influence their fate, but the specificresidues responsible for this phenomenon were notidentified (16).

The identify of the proteins that mediate the posten-docytotic sorting of GPCRs are just beginning to berevealed. EBP50/NHERF and N-ethylmaleimide-

sensitive factor have both been implicated as beinginvolved in the DSLL-dependent recycling of GPCRs(19, 20), and recent experiments conducted with themDOR and the thrombin receptor have identified twoproteins, sorting nexin 1 (40) and GPCR-associatedsorting protein (41) as proteins that participate in thesorting of internalized GPCRs to the lysosomes. Animportant future challenge for us will be to identify theproteins that sort the internalized LHR to a degrada-tion or a recycling pathway. This question is activelybeing investigated by searching for cellular proteinsthat show preferential binding to the C-terminal tails ofthe rLHR and hLHR in a GT-dependent manner.

MATERIALS AND METHODS

Plasmids and Cells

The preparation and characterization of expression vectorsfor the myc-hLHR-wt, myc-rLHR-wt, rFSHR-wt, and the FFLchimera have been described (24, 30, 42, 43). The differentmutants of the myc-rLHR (Table 1) and the FFL chimera usedhere were constructed by standard PCR strategies. Expres-sion vectors for Rab5a-GFP and procathepsin D-GFP weregenerously donated by Drs. Phil Stahl (Washington Univer-sity, St. Louis, MO) and Jonathan M. Backer (Albert EinsteinCollege of Medicine, Bronx, NY), respectively. An expressionvector coding for the mDOR modified to contain an HA-tag atthe N terminus was generously provided by Dr. Yi Ping Law(University of Minnesota, Minneapolis, MN). A mutant of theHA-mDOR (designated HA-DOR/hLHR) in which the 6 C-terminal residues of the DOR (GGGAAA, see Fig. 1) wereremoved and replaced with the 17 C-terminal residues of thehLHR (LHCQGTALLDKTRYTEC, see Fig. 1) was also con-structed using standard PCR strategies.

Human embryonic kidney 293 cells and 293T cells weremaintained in DMEM containing 10 mM HEPES, 10% new-born calf-serum, and 50 �g/ml gentamicin (pH 7.4). Cellswere plated in gelatin-coated 35-mm wells and transientlytransfected with 0.5 �g of plasmid DNA, using the calciumphosphate method of Chen and Okayama (44), when 70–80% confluent. After an overnight incubation with the trans-fection mixture, the cells were washed and used 24 h later.The preparation and properties of 293L(wt-1) cells, a clonalline of 293 cells stably expressing the rLHR-wt at a highdensity, have been described (45–47). Additional clonal linesof 293 cells stably expressing the myc-rLHR-GT mutant,designated 293Lmyc(GT-1), were obtained by selection ofthe transfected cells with 700 �g/ml of G418 as describedelsewhere (33, 45).

Rate of Internalization of hCG

Transiently transfected cells were incubated with a subsatu-rating concentration (�0.5 nM) of 125I-hCG at 37 C and thesurface bound and internalized radioactivity were measuredas a function of time after hormone addition. The surfacebound and internalized radioactivity were measured using abrief exposure of the cells to an isotonic pH 3 buffer (48), andthe half-times of internalization were calculated from theslope of the line obtained by plotting the internalized radio-activity against the integral of the surface-bound radioactivityusing at least five different data points collected at 10-minintervals after the addition of 125I-hCG (43, 49).

Galet et al. • Sorting of the Internalized LHR Mol Endocrinol, March 2003, 17(3):411–422 419

Fate of the Internalized hCG-LHR Complex

Transiently transfected cells were allowed to internalize 125I-hCG during a 2-h incubation at 37 C with a saturating con-centration of hormone (�50 nM). After washing to remove thefree hormone, the surface-bound 125I-hCG was released by abrief exposure of the cells to an isotonic pH 3 buffer (10, 48,49). This was defined as t � 0, and the cells (which nowcontain only internalized 125I-hormone) were incubated for anadditional 2 h at 37 C. At this time the medium was saved andthe cells were washed with cold medium, and they werebriefly exposed again to the isotonic pH 3 buffer to releaseand measure any of the internalized hormone that had recy-cled back to the surface. The acid-stripped cells were solu-bilized with NaOH to measure residual radioactivity thatremained internalized. Finally, the saved medium was pre-cipitated with 10% trichloroacetic acid to determine theamount of degraded and undegraded 125I-hCG released (10,48, 49).

Confocal Microscopy

Cells were plated in eight-chamber coverslip culture vesselscoated with polylysine (BioCoat from Becton Dickinson,Franklin Lakes, NJ). They were cotransfected (in a total vol-ume of 400 �l) with 100 ng of the appropriate myc-LHR orHA-mDOR constructs, and 8 ng of Rab5-GFP or cathepsinD-GFP using the methods described above. Two days aftertransfection, the cells expressing the LHR constructs wereincubated with or without hCG (52 nM) for 2 h at 37 C and thecells expressing the DOR constructs were incubated withDADLE (10 �M) for 30 min at 37 C. The medium was removed,and the cells were washed twice with PBS (137 mM NaCl; 2.7mM KCl; 1.4 mM NaH2PO4; 4.3 mM Na2HPO4, pH 7.4) andfixed during a 30-min incubation at room temperature with4% paraformaldehyde (dissolved in PBS). The fixed cellswere washed twice again and then incubated for 1 h at roomtemperature with PBS containing 50 mg/ml BSA. This solu-tion was removed, and the cells were incubated for anotherhour at room temperature with a 1:100 dilution of the anti-myc monoclonal antibody (9E10) or an anti-HA monoclonalantibody (12CA5) dissolved in PBS containing 5 mg/ml BSA.After washing four times with PBS, the cells were incubatedfor another hour at room temperature with a 1:2000 dilution ofCY5-labeled antimouse IgG. Finally, they were washed threeto four times with PBS, dried, and mounted in Vectashieldmounting medium (Vector Laboratories, Inc., Burlingame,CA). The CY5-labeled LHR and the arrestin-3-GFP were vi-sualized with a Bio-Rad Laboratories, Inc. (Richmond, CA)confocal microscope at the Central Microscopy Facility ofThe University of Iowa.

Phosphoaminoacid Analysis

Phosphorylated receptors were immunoprecipitated from293L(wt-1), 293Lmyc(GT-1) cells or from 293T cells tran-siently expressing the myc-hLHR-wt. The choice of stablytransfected or transiently transfected cells as a source ofrLHR and hLHR was based entirely on receptor abundance(18). Cells were metabolically labeled with 32P (400 �Ci/ml)for 3 h and stimulated with a saturating concentration ofagonist (�50 nM) for 15 min (cells expressing the rLHR-wt orrLHR-GT) or 60 min (cells expressing the hLHR-wt) as de-scribed elsewhere (28–30, 33, 47). The rLHR-wt expressed in293L(wt-1) cells is not myc-tagged and was immunoprecipi-tated with a polyclonal antibody to the rLHR as describedelsewhere (33, 50). Because the rLHR-GT expressed in293Lmyc(GT-1) cells and the hLHR expressed in transientlytransfected cells are both tagged with the myc epitope (seeabove), these receptors were immunoprecipitated with the9E10 antibody as described before (30).

The immunoprecipitates were bound to either protein Aagarose (for the polyclonal antibody) or to protein G-agarosefor the monoclonal antibody, the beads were washed exten-sively (30, 33, 50), and the immune complexes were elutedduring a 15-min incubation with 1 M acetic acid. The eluateswere dried under a stream of nitrogen and phosphoaminoacid analysis of the immunoprecipitated and eluted proteinswas carried out using a HTLE-7000 electrophoresis systemas described by Van der Geer and Hunter (51). Briefly, theeluted proteins were hydrolyzed in 6 N HCl for 1 h at 110 Cand dried using a Speed Vac with 4 �g of phosphoamino acidstandards (phosphoserine, phosphothreonine, and phospho-tyrosine). The dried samples were resuspended in 20 �l of2.5% (vol/vol) formic acid, 7.8% (vol/vol) acetic acid (pH 1.9)and spotted on a thin-layer chromatography plate (EM Sci-ence, Gibbstown, NJ). The samples were electrophoresed inthe first dimension in the same pH 1.9 buffer at 1500 V for 20min. The plate was dried, turned 90 degrees counterclock-wise, and electrophoresed in the second dimension in 5%(vol/vol) acetic acid, 0.5% (vol/vol) pyridine (pH 3.5) at 1300 Vfor 16 min. The plate was dried for 45 min, sprayed with 0.5%ninhydrin, and heated in an oven at 65 C for 10 min. The32P-labeled phosphoamino acids were detected by autora-diography and identified by matching with the standards.

Receptor Down-Regulation

293T cells that had been transiently transfected with themyc-rLHR-wt, myc-rLHR-GT, or myc-hLHR-wt were washedtwice with assay medium (Waymouth’s MB752/1 supple-mented with 1 mg/ml BSA; 20 mM HEPES; and 50 �g/mlgentamicin, pH 7.4). Some cells were saved on ice and pro-cessed immediately (t � 0 samples), whereas others wereincubated in 1 ml of warm assay medium containing a satu-rating concentration (�50 nM) of hCG for 6 h at 37 C. At thedesired time, the cells were placed on ice and lysed (52). Themyc-tagged LHR were immunoprecipitated with the 9E10antibody and the immunoprecipitates were resolved on so-dium dodecyl sulfate gels and electrophoretically transferredto polyvinylidene difluoride membranes as described else-where (52, 53). The antibody conjugated to horseradish per-oxidase, and the proteins were finally visualized and quanti-tated using the Super Signal West FEMTO MaximumSensitivity system of detection from Pierce Chemical Co.(Rockford, IL) and a Kodak (Rochester, NY) digital imagingsystem as described elsewhere (52). This digital image cap-ture system is set up to alert us when image saturation occursand to prevent us from measuring the intensity of suchimages.

The immunoprecipitation/immunoblot analysis describedabove and illustrated in Fig. 3 results in the identification ofthe mature LHR present at the cell surface (�85 kDa), anintracellular LHR precursor with a molecular mass of approx-imately 68 kDa, and an approximately 165-kDa aggregate ofthe 68-kDa precursor (18). Because only the cell surface LHRis expected to change as a result of agonist activation, weused the intensity of the 68-kDa band to correct for loadingand variability in receptor expression. Thus, to calculatedown-regulation (see Table 2) the amount of cell surfacereceptor (i.e. 85 kDa) was divided by the amount of receptorprecursor (i.e. 68 kDa) and the corrected data from the cellsincubated with hCG for 6 h were expressed as the percent-age of the corrected data from the cells that had hCG addedbut were processed immediately after hormone addition.

Hormones and Supplies

Human kidney 293 cells and the 9E10 hybridoma cell linewere obtained from the American Type Culture Collection(Manassas, VA). The 9E10 cells were used by the HybridomaFacility of the Cancer Center of the University of Iowa toprepare a concentrated supernatant containing the 9E10 an-

420 Mol Endocrinol, March 2003, 17(3):411–422 Galet et al. • Sorting of the Internalized LHR

tibody. The 9E10 antibody coupled to horseradish peroxi-dase was purchased from Roche Molecular Biochemicalsand the 12CA5 monoclonal antibody to the HA epitope wasfrom Roche. The CY5-labeled secondary antibody was fromThe Jackson Laboratory (Bar Harbor, ME). DADLE was fromSigma (St. Louis, MO). Human kidney 293T cells are a deriv-ative of 293 cells that express the Simian virus 40 T antigen(54) and were provided to us by Dr. Marlene Hosey (North-western University, Chicago, IL). Purified hCG (CR-127,�13,000 IU/mg) and hFSH (AFP-5720D, prepared from hu-man pituitaries) were purchased from the National Hormoneand Pituitary Agency (NIDDK, NIH, Bethesda, MD) and puri-fied recombinant hCG and hFSH were provided by Ares-Serono (Randolph, MA).1 125I-hCG was prepared as de-scribed elsewhere (55). Partially purified hCG (�3000 IU/mg)was purchased from Sigma, and it was used only for thedetermination of nonspecific binding. Partially purified equineFSH was kindly donated by Dr. George Bousfield (WichitaState University, Wichita, KS). Cell culture medium was ob-tained from the Media and Cell Production Core of the Dia-betes and Endocrinology Research Center of the Universityof Iowa. Other cells culture supplies and reagents were ob-tained from Corning, Inc. (Corning, NY) and Invitrogen (Carls-bad, CA), respectively. All other chemicals were obtainedfrom commonly used suppliers.

Acknowledgments

We thank Dr. Deborah Segaloff (The University of Iowa,Iowa City, IA) for her comments on this manuscript, Dr.Marlene Hosey (Northwestern University, Chicago, IL) for293T cells, Dr. Phil Stahl (Washington University, St. Louis,MO) for the Rab5a-GFP expression vector, Dr. Jonathan M.Backer (Albert Einstein College of Medicine, Bronx, NY) forthe procathepsin D-GFP expression vector and Dr. Ping-YeeLaw (University of Minnesota, Minneapolis, MN) for the HA-mDOR-wt expression vector. We also thank Professor Ma-satomo Mori (First Department of Internal Medicine, GunmaUniversity, Gunma, Japan) for his support. Lastly, we thankAres Serono (Randolph, MA) for providing us with the purifiedrecombinant hCG and hFSH and the initial hLHR plasmidused in these experiments and Dr. George Bousfield (WichitaState University, Wichita, KS) for the partially purified equineFSH.

Received April 30, 2002. Accepted November 20, 2002.Address all correspondence and requests for reprints to:

Dr. Mario Ascoli, Department of Pharmacology, 2-319B BSB,51 Newton Road, The University of Iowa, Iowa City, Iowa52242-1109. E-mail: mario-ascoli@uiowa.edu.

This work was supported by NIH Grants CA-40629 (toM.A.) and CA-57539 (to N.L.W.). The services and facilitiesprovided by the Diabetes and Endocrinology Research Cen-ter of the University of Iowa (supported by NIH Grant DK-25295) are also gratefully acknowledged.

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