A Luteinizing Hormone Receptor with a Severely Truncated ...

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1723-1728, 1993. (0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.. A Luteinizing Hormone Receptor with a ...
THEJOURNALOF BIOLOGICAL CHEMISTRY (0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 268, No. 3, Issue of January 25, pp.Printed 1723-1728, 1993 in U.S.A.

A Luteinizing Hormone Receptorwith a Severely Truncated Cytoplasmic Tail (LHR-ct628) Desensitizes to the Same Degree as the Full-length Receptor* (Received for publication, July 9, 1992)

Xi ZhuS, Thomas GudermannSQ, Marie1 BirnbaumerS, and Lutz BirnbaumerSliII From the Departmentsof $Cell Biology and (Molecular Physiology and Biophysics and the 11 Division of Neurosciences, Baylor College of Medicine, Houston, Texas 77030

ties. Like rLHR, the mature mLHRis predicted to have 674 amino acids subdivided into two major domains: a hormone binding domain, formed of an extracellular amino-terminal portion that comprises approximately 294 amino acids encoded in 10 exons (Tsai-Morriset d., 1991; Koo et d., 1991), and a signal transducing carboxyl-terminal portion thatcomprises the remaining380 amino acids encoded in a single11th exon (see Fig. 1).This second domain has the typical structural motifs of a G protein-coupled receptor which predict seven transmembrane domains, three extracellular and three intracellular loops, andanintracellular carboxyl terminus (Rodriguez and Segaloff, 1990). The latter is predicted to be of 69 amino acids containing 2 vicinal cysteines (Cys6” and Cys6“) a t positions -53 and -54 from the carboxyl terminus. These cysteines may be palmitoylated in analogy to Cys3” and Cys323of rhodopsin and C Y S ~in~ the ’ &adrenergic receptor and provide an eighth membrane anchor (O’Brien and Zatz, 1984; Ovchinnikov et al., 1989; O’Dowd et al., 1989). Binding of LH and its functional homologue hCG to the LH receptor elicits a minimum of three responses: stimulation of adenylyl cyclase activity (Marsh et al., 1970) presumably through activation of G, (Kirchick et al., 1983) which leadsto an elevationof intracellular CAMPlevels, stimulation of PIP2specific phospholipaseC, either through activation of aG protein of the G, class or by some other means which leads Cloning of the rat and porcine luteinizing hormonereceptor to a rapid elevation of intracellular Ca’’ levels (Davis et al., (LH receptor or LHR’) gave access to their primary amino 1984 and 1987; Gudermann et al., 1992a and 1992b), and a acid sequences (McFarland et al., 1989; Loosfelt et al., 1989) presumably secondary desensitization, whereby the stimulaand has led several laboratories to initiate structure-function tory signalsof the receptor are rapidly attenuated (Lamprecht studies to define the features that underlie hormonebinding, et al., 1973; Hunzicker-Dunn and Birnbaumer,1976). signal transduction anddesensitization. Using thenucleotide The molecular basis of desensitization has been studied sequence reported for the rat LH receptor (rLHR) as a basis, extensively for the @-adrenergic receptorfor which it hasbeen we cloned its highly similar murine homologue (mLHR) and shown to involve the participation of both phosphorylation characterized both its predicted amino acid sequence (94% by a kinase such as CAMP-dependent protein kinase and (3identical to thatof rLHR) and some of its signalling proper- adrenergic receptor kinase, a memberof a familyof G proteincoupled receptor kinases, and a segregation or internalization * This work was supported in part by Grants HD-09581 and HLprocess which may or may not depend on prior phosphoryla45198 (to L. B.), DK-41244 (to M. B.), and HD-07485 (to theBaylor tion. This has as itsconsequence a physical uncoupling from College of Medicine)from the National Institutes of Health. The costs of publication of thisarticle were defrayed in part by the the G,-adenylyl cyclase system (Lohse et al. (1990); reviewed payment of page charges. Thisarticlemusttherefore be hereby in Hausdorff et al. (1990)) and, in time,collection in endosomarked “aduertisement” in accordance with 18 U.S.C. Section 1734 mal structures and degradation (von Zastrow and Kobilka, solely to indicate this fact. 1992). As shown by Valiquette et al. (1990), desensitization of § Recipient of a fellowship from the German Research Council (Deutsche Forschungsgemeinschaft). Present address: Inst. of Repro- receptors may not only involve serines and threonines but ductive Medicine, University of Munster, D-4400 Munster, Germany. also tyrosines. Althoughthis may be different for other recepThe abbreviations used are: LHR, LHreceptor; G protein, one of tors, most if not all of the @-adrenergic receptorkinase phosa family of signal transducingheterotrimericguanine nucleotidephorylation sites of the @-adrenergic receptor are located in binding proteins; hCG, human chorionic gonadotropin; LH, luteinizthe carboxyl-terminal tail of the molecule, as is the tyrosine ing hormone; PCR, polymerase chain reaction; RT,reverse transcriptase; RT-PCR, reverse transcription followed by polymerase chain that was found to affect the desensitizing process as a whole. As shown below, the murine LHreceptor, stably expressed reaction; MMLV, murine moloney leukemia virus; DTT, dithiothreitol; DEPC, diethyl pyrocarbonate. in murine L cells, undergoes a desensitization reaction which

The wild type murine luteinizing hormone(mLH) receptor, which in its mature form is predicted to be a protein of 674 amino acids (mLHR),and anartificially mutated form lacking the last 46 amino acids (mLHRct628) were stably expressed in murine L cells. Both forms stimulated adenylyl cyclase and underwent rapid desensitization. The mutation removed 1 tyrosine, 2 threonines, and 6 serines from the receptor. The results indicate that none of these potential phosphorylation sites participates in either adenylyl cyclase stimulation or receptordesensitization. Our resultswith themLHR-ct628(carboxyl-terminal amino acid sequence CCKHRAEL) differ from those reported recently for the essentially identically mutated rat LHR that lacks the last 43 amino acids (rLHRct631 with carboxyl-terminal amino acid composition CCKRRAELYRR).This 43-amino acid truncation was described to have the effect of preventing hormoneinduced desensitization. While the reasons for the discrepant results are not known, our results do not support the proposal for a participatory role of the extreme carboxyl terminus of the receptor in its desensitization.

1723

1724

Normal Desensitizationof a TruncatedLH Receptor

t

...

mLHR-ct628 I Sequence of pAGA derived from pGEM-SZf(-)

--f

GCT GAA CTT TAG CATG CAAGCTTGAGTATTC TATAGTGTCACCTAAAT-3'

Glu. . . Ala aa codon number

Leu S t o p SphI 628

SP6 promoter

(Eq. 1)

that ifby chancethe truncated receptor would still desensitize,The predicted composition ofthe L~R-~tfj28/pKNH junction in p ~ this strategy could lead to a reduction of the complexity of LHR-ct628 is as follows.

mLHR-ct628 1 pKNH 20 40 . . . GCT GAA CTT TAG AGCTTGAATT CACTCCTCAG GTGCAGGCTG CCTATCAGAA t

aa

Glu

. . . Ala

codon number

--f

Leu S t o p

EcoRI

(Eq. 2)

628

t h e molecule and facilitate the subsequent search for the molecular parameters that define the desensitizing response of this receptor. Work of Segaloff and collaborators, first published in abstract form (Rodriguez et al., 1991), on effects of carboxyl-terminal truncations on receptor expression indicated that inclusion of the double cysteineat positions 621 and 622interfereswithreceptormaturation and leads t o intracellular accumulation of receptor protein. We thus engineered a mutant, mLHR-ct628, that excluded all of the potentialphosphorylationsitesdownstream of t h e double cysteines and begun studying its signalling propertiesas seen i n clonal cells that express it i n a stable constitutive manner. While we were in the processof cloning cells expressing the murine LHR truncated at position 628, Segaloff and collaborators (Sinchez-Yagiie et al., 1992) publishedthe results from a very similar study. They truncatedthe rat LHR at position 631 and found that this mutation both enhanced the intrinsic activity of the receptor and prevented hCG-induced desensitization. Although we expected to confirm their finding, we found the desensitizing response of the mLHR-ct628 to be indistinguishable fromthat of the wild type. EXPERIMENTALPROCEDURES

cDNAs, Eukaryotic Expression Vector, and Cell Transfections-For the present experiments the wild type form of the full length NcoI(-l)/NcoI(+2563) murine LHR cDNAwas excised from the replicative form of M13 mp18 (Gudermann et al., 1992a), rendered blunt-ended with the Klenow fragment of Escherichia coli DNA polymerase, and ligated in gel into the eukaryotic expression vector pKNH (Nukada et al., 1987; Takeshima et al., 1989) that had been cut with HindIII, rendered blunt-ended with Klenow fragment of DNA polymerase and dephosphorylated with calf intestinal phosphatase, to give plasmid pK-LHR (also pK-LHR-wt). Calling nucleotide 1 the A of the ATG initiation codon at amino acid -26 of the mature LHR, themutant of the murine LHR cDNA truncated by 598 nucleotides was created by excising an EspI (unique site in mLHR cDNA at nucleotide +1925)/SphI (unique sitein polylinker of pAGA) fragment from pAGA-mLHR (Gudermann et al., 1992b; Sanfordet al., 1991) and ligating into its place a preannealed synthetic double stranded nonphosphorylated EspI/SphI fragment of 38 base pairs plus 5' Espl and 3' SphI overhangs, that restores nucleotide 1925 to

The nucleotide composition at thesejunctions was confirmed by direct sequencing (Sanger et al., 1977) using double stranded plasmid DNA as template. The expression vectors containing both LHRs, pK-LHR with an insert length of2563 base pairs and pK-LHR-ct628 with an insert length of 1965 base pairs, were transfected into Ltk- cells by the calcium phosphate/glycerol shock method (Graham and Van der Eb, 1973).G418-resistant transformants were selected, replicated into 96well microtitration plates, and tested in situ for acquisition of LHsensitive adenylyl cyclase activity as described (Birnbaumer et al., 1990; Themmen et al., 1993). pK-LHR gave rise to colonies denoted as pKx LHR colonies and pK-LHR-ct628 gave rise to colonies denoted as pKx LHR-ct628 colonies. Cells from replicas of those in positive wells (primarytransformants) were expanded and tested further. Several primary clones were obtained for each of the receptor types, and some of these were subjected to clonal selection by limiting dilution to isolate clonal cell lines, denoted as pKx/y LHR clones and pKx/y LHR-ct628 clones. Experiments reported below were carried out with clones pK5/27 LHR cells and pK33/12 LHR-ct628 cells. Cell Growth, Treatments, AdenylylCyclaseActivityAssays, and Determination of hCG Binding to Intact Cells-Cells were grown as described previously in minimal essential cy medium and 10% fetal calf serum containing penicillin and streptomycin (Gudermann et al., 1992a)supplemented with 400 pg/ml of G418 (GIBCO) and harvested from 100- or 150-mm plates when they reached 75-85% confluence. For adenylyl cyclase assays, cells were rinsed at room temperature with a balanced salt solution (Gudermann et al., 1992a), scraped off the plates with the aid of a rubber policeman into ice-cold balanced salt solution, collected by centrifugation, and resuspended in ice-cold homogenization medium consisting of 27% (w/v) sucrose, 1 mM EDTA, and 20 mM Na-Hepes, pH 7.5, at a ratio of 1 ml of medium per cells from a 150-mm dish. Homogenization, centrifugation through discontinuous sucrose density gradient, isolation of membranes, and adenylyl cyclase assay conditions were as described (Gudermann et al., 1992a). Hormone binding to intact cells was performed on cells grown in 12-well tissue culture plates to a density of 0.8 to 1.0 X lo6 cells/well. Cells were rinsed a t 4 "C with Dulbecco's modifiedphosphate buffered saline (GIBCO) supplemented with 0.2%glucose and 1% bovine serum albumin, and binding reactions were initiated by adding to each well 0.7 ml of the same medium containing the appropriate dilutions of lz5I-hCG.The incubations were carried out for 15 h at 4 "C. Nonspecific binding was determined under the same conditions in the presence of 10 Ng/ml unlabeled hCG.At the end of the incubation the wells were quickly rinsed three times with the same

Normal Desensitization

Truncated of a

LH Receptor

1725

medium without hormones and 1.0 ml of 0.1 N NaOH was added to 10min at room temperature followed by 90min a t 37 "C. The extract the bound radioactivity. The plates were then brought to room reactions were stopped by heating to 95 "C for10 min. For amplificationof DNA by the polymerase chain reaction (PCR), temperature, incubated for 30 min a t 37 "C, and the NaOH solutions from the wells were transferred to 1 2 x 75-mm test tubes. Radioac- 2.5 pI of the first strand cDNA synthesis reactions described above or 2.5 pl with 2.5-5 pg/ml of pK-LHR-wt or pK-LHR-ct628plasmid tivity was determined using a y-spectrometer. In separate assays it was shown that all of the specifically bound DNA, were mixed with 97.5 pl of PCR mix consisting of 10 pl of 10 counts could be removed from the cell-containingwells by two 2-min x PCR buffer (1 X PCR buffer = 10 mM Tris-HC1, pH 9.0, 50 mM washes with 2 ml of an ice-cold solution of 5 mM acetic acid in 135 KCl, 1.5 mM MgCI,, 0.1% Triton X-loo), 0.7 pl of 25 mM dNTPs, 2 p1 each of 50 p~ (100 pmol) sense and 50 p M (100 pmol) antisense mM NaCl and 2.5 mM KC1, pH 3.7 (not shown). Based on this, the radioactivity recovered inthe wells was ascribed to cell surface primers, 0.5 pl (2.5 units) of Taq polymerase (Promega), and 82.3 pl of H,O. The samples were overlaid with 75 pl of mineral oil and receptor binding. Desensitizing Treatments-Desensitization of LHR cells by hCG amplified in a thermocycler. The PCR program heated the samples was performed in150-mmplateswith cells growing in 20 ml of to 96 "C for 7 min, cycled the mixtures 30 times through 96 "C for40 s, 60 "C for 40 s, and 72 "C for 120 s, followed by a final step a t 72 "C minimal essential a medium/lO% fetal calf serum plus additives as were analyzed by 1.2% listed in theprevious paragraph. Reactions were initiated by addition for 12 min. The reactionproducts(20pl) of aliquots of 2 mg/ml hCGto thegrowth mediumto give the indicated agarose SeaKem (FMC Bioproducts)gel electrophoresis in 0.5 X Tris concentrations. After incubation of the cells a t 37 "C for 30 min, the borate/EDTA buffer (Maniatis et al., 1982) at 100 V for 2 h in the treatments were stopped by rinsingthe cellsonce with ice-cold presence of 1pg/ml ethidium bromide. Sense primer (primer mLHRDulbecco's modified phosphate-buffered saline, followed by two 2- ct: 5"CCTCAATGCA GTGGCCTTTG-3') corresponds to nucleomin washes with an ice-cold solution composed of either 50 mM tides 1610-1629 of the murine LHRcDNA (Gudermann etal., 1992a). glycine in 100 mM NaCl, pH 3.0, or 5 mM acetic acid in 135 mM NaCl and 2.5 mM KC1, pH 3.7, followed by two rinses with ice-cold DulRESULTS becco's modified phosphate-buffered saline. Controlcells to which no hCG was added were washed in the same manner. Cells were then Our previous studies were performed with a clonal L cell harvested, and membranes were prepared for adenylyl cyclase assay line obtained by cotransfection of plasmid p91023(B)carrying as described above. the LHR cDNA and plasmid pHSV-106 carrying the thymiAnalysis of LHR and LHR-ct628 Transcripts in pK5/27 LHR and pK33/12 LHR-ct628 Cells-For isolation and analysis of LHR and dine kinaseselection marker. Transformedcells were isolated LHR-ct628mRNA,150-mmplateswith cells grown as described by growing in hypoxanthine/aminopterin/thymidine medium. The resulting cells were LHR'ltk' (Gudermann et al., 1992a). above were removed from the CO, incubation, growth medium was aspirated and the cell layer was rinsed once with 10 ml of 150 mM For the present studies we obtained new cell lines by transNaCI, 20 mM Tris-HC1, pH 7.4, and then lysed by overlaying 6.6 ml fectingLtk-cells withplasmidpKNHcarryingboththe of 4 M guanidinium isothiocyanate, 20 mM sodium acetate, 0.1 mM cDNA to be expressed (wild typeLHR or the carboxylDTT, 0.5% Sarkosyl. Cells and cell debris were detached from the neomycin resistplate, and the lysatewas passed four times througha gauge 20 needle terminally truncated ct628 LHR) plus the ance marker. Transformed cells were isolated in G418-cont o shearthe DNA.RNA was then isolatedfrom thislysate by growthmedium. Theresulting cells (pK centrifugation through a 3.0-ml cushion of 5.7 M CsCl, 0.1 mM EDTA tainingnormal as described by Chirgwin et al. (1979). The resultingpellet was taken clones) are LHR'/tk- or LHR-ct628+/tk-. Table I summarizes u p in 360 p1 of 10 mM Tris-HCI, pH 7.4, 1 mM EDTA, and 1%SDS, the result from these transfections. precipitated in a microcentrifuge tube by addition of40 pl of 3 M The average stimulation by hCG of the hCG-responsive sodium acetate, pH 5.2, and 1 ml of ethanol (30 min at -70 "C), and primary cell clones expressing the LH receptor truncated at collected by a 10-min centrifugationat 4 "C. The pellet was taken up in DEPC-treated water, reprecipitated as above, taken up once more amino acid 628 (LHR-ct628), was 5.1 f 1.2 over basal, and did notdiffer significantly fromthat obtainedfor primary cell in DEPC-treated water to a concentration of approximately 1 mg/ ml, and precipitated by addition of 3 volumes of 4 M sodium acetate, lines expressing the full length LHR under the control of the p H 6.0. The RNA was collected by centrifugation after chilling on ice same expression vector (6.7 f 2.3; Table I). We thus saw no for 60 min, washed oncewith 70% ethanol, air-dried, and resuspended evidencefor an enhancement of intrinsic activity of LHR i n 100 pl of DEPC-treated water. The presence of LHR transcripts in LHR and LHR-ct628 cell resulting from truncating its cytoplasmic tail at position 628 RNA was analyzedby first preparinga specifically primed first strand (Fig. 1).This differs from the property of the truncation at cDNA using murine moloney leukemia virus (MMLV) reverse tran- amino acid 631 (LHR-ct631) which was reported by Sanchkzscriptase and total RNA as template, and then amplifying the cDNA Yagiie et al. (1992) to have an enhanced intrinsicactivity. by polymerase chain reaction (RT-PCR). Prior touse, total RNAwas Clonal cell lines were obtained from primary transformants treated with DNase. For this, 12-15 pg RNA was treated in a final by limiting dilution.Clonal cell lines expressing the wild type volume of 100 pl with 2 pl FPLC-pure DNase I (Pharmacia LKB of pKNHratherthan Biotechnology Inc.), 2 p1 of RNAguard ribonuclease inhibitor (Phar- L H receptor underthecontrol macia), 5 p1of 0.1 mM DTT, 10 pl of 10 X buffer (1 X buffer = 50 p91023(B) did not differsignificantly in terms of relative mM Tris-HC1, pH 7.5, 10 mM MgCl,), and DEPC-treated water to stimulation of adenylyl cyclase activity by LH or hCG or the volume.After an incubation of 10 min at 37 "C the reaction was EC50of hCG action. Thus, activation of adenylyl cyclase by stopped by heating to 68 "C for 3 min, and the mixturewas extracted hCG of LHR clone pK5/27 was half-maximal at 0.3 nM, as once with phenol/chloroform. The RNA in the aqueous phase was precipitated by addition of 10 pl of 3 M sodium acetate, pH 5.2, and TABLEI 2.5 volumes of 100% ethanol, chilled a t -70 "C for 1 h, collected by centrifugation, washed twice with 70% ethanol, dried, andresusSummary of hCG responsiveness of adenylyl cyclase in primaryG418pended in 25 p1 of DEPC-treated water. resistant clones obtained upon transfecting Ltk- cells with pKNHFor reverse transcription, duplicate aliquotsof 10 pl of the DNaseLHR-wt ILHR-DK cells) and D K N H - L H R - cILHR-ct628 ~~~~ cells) treated RNA were placed into 1.5-ml microcentrifuge tubes, heated Plasmid transfected t o 68 "C for 5 min, and rapidly cooled to 0 "C. To each tube were added 9 pl of medium containing 0.8 p1 of 25 mM dNTPs, 4 pl of 5 X MMLV buffer (1X MMLV buffer = 50 mM Tris-HC1, p H 8.3, 75 mM 48 66 KCl,and 3 mM MgCl,), 0.5pl of 0.1 M DTT, 1 p1 RNAguard Number of G418-resistant colonies picked (3"CTTAAGT(Pharmacia), and1p1 of 50 p~ (50 pmol) primer pK2 Number stimulatedby hCG 15 (31%) 21 (31%) GAG GAGTCCACGT-5' complementary to pKNHnucleotides 6-25 more than 1.9-fold in the scheme shown above). The mixture was heated once more to 3 (6%) 4 (6%) 68 "C for 5 min then allowed to cool slowly to room temperature. One Number stimulated by hCG from 1.4- to 1.9-fold tube (plus RT)received 1pl (200 units) of MMLV reverse transcrip5.1 f 1.2 6.7 & 2.3 tase (Bethesda Research Laboratories), while another (noR T control) Mean stimulation by hCG of >1.9-fold group (S3.D.) received 1 pl of DEPC-treated water. The tubes were incubated for

Normal Desensitization

1726

of a Truncated LH Receptor

b

'

4361--iil 23222027-,

ct628 ct631

-LHR nonspecific -LHR-Ct628

564-

J

* 674 FIG. 1. Model of structure of the LH receptor and illustration of place and nature of the mutations relevant to the present report. Mutant ct628 terminates with amino acid 628 (L)

RT

. +. . . . . -

f -

FIG.2. RT-PCR analysis of the type of LHR expressed in LHR cells. Truncation of the LHRcDNA to give LHR-ct628 cDNA

and mutant ct631 terminates with amino acid 631 ( R ) .The lengths of lines are proportional within 10% to the lengths of polypeptide predicted a shortening of the PCR product from 979 to 381 base pairs, chains. Unique structural features of the LH receptor in relation to i.e. by 598 base pairs. For experimental details see "Experimental other G protein-coupled receptors outside the subfamily of the gly- Procedures" and text. and - RT denote addition or not of reverse coprotein hormone receptors (LH, follicle-stimulating hormone, and transcriptase during the synthesisof the first strandcDNA. thyrotropin)arethe large extracellularamino-terminal region of LHR, which is shown folded to indicate imperfect 25-aminoacid-long repeats proposed by McFarland et al. (1989), although a more recent corresponds to 14,400 and 6,000 sites per cell. The LHR-wt study recognizes only eight such repeats (Koo et al., 1991), and a and LHR-ct628 receptors boundhCG with Kd values of 150 relatively short third intracellular loop of only 25 amino acids. A and 350 PM, respectively. The ECso for adenylyl cyclase stimdisulfide bond (-OO-) ispostulated between the first and second ulation by hCG in partially purified membranes was 270 pM extracellular loops based on the mutational studies of Khorana and in pK5/27 cells expressing wild type LHR (range 250-300 colleagues with rhodopsin (Karnik et al., 1988), and the conservation LHR-ct628 of cognate cysteinesin almost all G protein-coupled receptors. Folding PM) and 540 PM forpK33/12cellsexpressing (range 500-600) (Fig. 4). Thus, the ratio ECso/Kd for the of amino and carboxyl termini is arbitrary. The anchoring of the carboxyl terminus is patterned after that of rhodopsinwith two interaction of hCG with receptor and activation of adenylyl palmitoyl anchors (O'Brien andZatz, 1984; Ovchinnikov et al., 1988) cyclase was similar for the two cell lines expressing the two and thatof the P-adrenoreceptorwith one palmitoyl anchor (O'Dowd forms of t h e L H R1.8 for the wild type receptor in clone pK5/ et al., 1989). Asterisks indicate the possible sites of phosphorylation; 27 and 1.6 for the truncated form of the receptor. Furtherthe black and gray pattern of the 14 repeats of the extracellular domain is symbolic and does not represent true animoacid sequence more, since 50% of adenylyl cyclase stimulation required a similar (60-65%) proportion of the receptors to be occupied, similarities of the imperfect repeats.

+

compared to 0.2 nM in the LHR 11/6cell line in which LHR expression is under the control of the p91023(B) expression vector, and maximal increases in both cell types are 6-8-fold above control activity as measured in the presence of 0.1 mM ATP, 5 mM MgC12, 20 PM GTP, and 1 mM EDTA. The effect of truncating the LHR at position 628was studied by comparing the properties of the hCG-stimulated activity and desensitizing responses of two clonal cell lines: pK5/27 which expresses the full length wild type LH receptor and pK33/12 which expresses thetruncated form of the receptor. RT-PCR analysis of LHR transcripts isolated from LHR pK5/27 and LHR-ct628pK33/12 cells confirmedthe identity of the receptor forms expressed in these cells (Fig. 2). Functional expression of the protein was assessed both by direct binding of hCG to intact cells and stimulation of adenylyl cyclase by increasing concentrations of hCG in homogenates and membranes. As shown in Fig. 3, the density of hCG-specific binding sites on thesurface of LHR and LHR-ct628cells was 24 and 10 fmol of hCG binding sites perlo6cells, respectively, which

this indicated that coupling between receptor occupancy and effect is relatively linear, and that the receptors were not being excessively overexpressed in relation to the G.-adenylyl cyclase effector system. The clonal pK5/27 LHR andpK33/12 LHR-ct628 cell lines differed by about %fold in the specific activities of their adenylyl cyclase systems as seen in homogenates measuring ) activity (not shown) aswell as forskolin (100 p ~ stimulated in membranes measuring basal and hCG-stimulated activities (Fig. 4). This difference was not unexpected for we have observed up to %fold differences in intrinsic adenylyl cyclase activity of clonal cell lines of the same type. Differences of this kindvery likely represent normal cell-to-cell fluctuations that either exist naturally prior to transfectionor which are brought out by the transfection andselection process. Both the cells with wild type LHR and the cells with the ct-628 form of the LHR appear to have not only a normal stimulatory reaction to hCG but also a normal desensitizing reaction to the hormone. Clone pK5/27 LHR cells and clone pK33/12 LHR-ct628 cells were exposed for 30 min a t 37 "C to 1 or 10 pg/ml hCG. These cells, and control cells that had received diluent, were then rinsed at acidic pH to remove

-ct628

ined

of a Truncated LH Receptor

Normal Desensitization

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I

FIG. 3. Saturation analysis of binding of hCG to intact pK5/27 LHR and pK33/12 LHR cells were plated into 12-wellplates and allowed to grow to close to confluence (0.97 x 106/well for LHR pK5/27 cells and 0.84 X 106/well for LHR-ct628 pK33/12 cells). After rinsing they were overlaid with 700 pl of medium containingincreasingconcentrations of hCG (3,346 cpm/fmol) for 15 h at 4 “C. The media were then removed, and bound Iz5I-hCG was then determined as described under “ExperimentalProcedures.”Incubations were in duplicate. Nonspecific, binding the presence of 10 pg/ml unlabeled hCG.

-200

I

0 C

r

3

a

10L - HR-~1828 c l opnKe 31 32 /

Total

-40

Specific

c

KdS35OpM Bmax=lo frnolllo~ce~ls

Y

-30

% m

-20 -10

Nonapacilic I

1.5 0 1.0

0.5 1251-hCG

2.0 A d d e d (nM)

I 2.50

L H R - c t 8 2 8 Membrane8

L H R - w t Membrane8

-0 4 6 8 10 lz51-hCG Bound (fmol/1O6cells) 2

19

30

1.-

Clone p K S I 2 7

1

E

0’

1’2

1’1

110 hCQ (-log

b

MI

A

;’

~~

1:

1’1

lb

b

co

hCQ (-109 M)

FIG. 4. Hormonal responsiveness and desensitization of L cells expressing the wild type LH receptor or a mutant receptor lacking the last 46 amino acids of its approximately 53-amino acid-long cytoplasmic tail. Cells were grown to close t o confluence and treated in situwith the indicated concentrationsof hCG for 30 min at 37 “C. After washing with glycine-based acid washingsolution, pH 3.0, to remove extracellularly bound hCG, the cells were harvested and homogenized. A partially purified membrane preparation was made in which adenylyl cyclase activity was measured as a function of hCG concentration. Vertical linesdenote concentration of hCG giving 50% of maximal stimulation, which are given in numerical terms for membranes from cells t o which no hCG was added during the treatment. Essentially the same results were obtained when acetic acid-based washing solution (pH 3.7) was substituted for the glycine-based solution. For further detailssee text and “Experimental Procedures.” unbound and surface bound hCG, harvested, and homoge- to trigger a desensitization response. By extension, the desennized as described under “Experimental Procedures.” Par- sitizing responsedoes not require the participation of the tially purified membrane fractions were prepared, and their missingcytoplasmic tail, includingseveral potential phoshCG-responsive adenylyl cyclase was determined as a func- phorylation sites: 6 serines, 2 threonines, and 1tyrosine. tion of hCG in the assay. Fig. 4 shows that thehCG-responsive Our results differ from and were not predicted by those adenylyl cyclase systems of both cells desensitized to equiva- presented in arecent report on the consequences of truncating lent degrees. Desensitization of the LHR is characterized by the LHR at position 631 rather than 628 (Sanchez-Yagiie et a loss of hCG-stimulated activity without a significant shift al., 1992). Cells expressing the ct631 receptor were somehow in the position of the dose-response curve for hCG (Fig. 4). Reduction in the concentration of hCG during the desensiti- prevented from desensitizing. The reasons for the different zation treatment from 10 pg/ml (0.23 PM) to 1 wg/ml (0.023 results are not understood. One, to us unlikely, possibility is p M ) resulted in a reduction of the desensitizing response in that themolecular aspects thatconfer a desensitizing response both cell lines. This indicated that the sensitivity to desensi- to the rat LHR (subject of the 631 truncation) differ from tization by hCG appears to be the same for cells with wild those of the murine LHR(subject of the 628 truncation). The two receptors are evolutionarily very close, as are thespecies, type and cells with the ct628 LHR. both rodents have almost identical reproductive characterisDISCUSSION tics, and one would not foresee an evolutionary difference in Our results indicate that an LH receptor lacking the last the desensitization mechanism,e.g. different choice of amino 46 amino acids is both able to stimulate adenylyl cyclase and acid to phosphorylate and uncouple the receptor from the G

1728

Normal Desensitization

of a Truncated LH Receptor

proteins. A second, to us more likely, possibility is that the 631 truncation was “unfortunate” in that it createda tyrosinyl-arginyl-argininecarboxyl terminus which, dueto its bulkiness and positive charge density, interfered nonspecifically with the desensitizing reaction. Since positively charged polypeptides are able to nonspecifically activate G proteins (cf. Antonelli et al. (1991)),the new arginyl-arginine carboxyl terminus may have also been responsible for the enhanced intrinsic activity of the LHR-ct631 which is absent from the LHR-ct628. Experiments reported in abstract form (Gudermann et al., 1992c) and to be described in detail elsewhere have shown that the40-50% desensitization of the hCG-stimulated activity we observe occurs without loss of more than 10-15% of the cell surface binding sites for hCG. This indicates that of the three classical mechanisms of receptor desensitization, phosphorylation by CAMP-dependent protein kinase, phosphorylation by a (Gprotein-coupled) receptor kinaseand rapid sequestration/internalization,the primary pathway(s) of desensitization operating on the LH receptor in L cells is phosphorylation by an as yet unidentifiedkinase. The present experiments should simplify the search for the putative site or sites of phosphorylation that lead to LH receptor desensitization. REFERENCES Antonelli, M., Olate, J., Allende, C. C., andAllende, J. E. (1991) Comp. Biochem. Physiol. B 99,827-852 Birnbaumer. M.. Hinrichs., V.., and Themmen. A. P. N. (1990) . . Mol. Endocrinol. ~4245-254 , ’ Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Bipchemistry 1 8 , 5294-5299 Davis, J. S., West, L. A,, and Farese, R. V. (1984) J. Biol. Chem. 2 5 9 , 1502815034 Davis, J. S., Weakland, L. L., Farese, R. V., and West, L. A. (1987) J. Biol. Chern. 262,8515-8521 Graham, F. L., and Van der Eb, A. J. (1973) Virology 5 2 , 456-467 Gudermann, T., Birnbaumer, M., and Birnbaumer, L. (1992a) J. Biol. Chem. 267,4479-4488

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