Follitropin (FSH) and a Phorbol Ester Stimulate the Phosphorylation of the FSH Receptor in Intact Cells*

Using a cell line stably transfected with the rat folli- tropin (FSH) receptor cDNA we demonstrate that the FSH receptor becomes phosphorylated when cells are exposed to FSH. Since binding of FSH to its receptor results in an increase in CAMP and inositol phosphate accumulation, we examined the potential involvement of protein kinase A and C in mediating receptor phos- phorylation. Stimulation of protein kinase A does not appear to be necessary because hFSH-induced receptor phosphorylation was minimally impaired in a cell line that overexpresses cAMP phosphodiesterase. Moreover, stimulation of the protein kinase A pathway with other agonists result in minimal phosphorylation of the FSH receptor. Stimulation of the protein kinase C with a phorbol ester did result in an increase in receptor phosphorylation, and down-regulation of the protein kinase C decreased, but did not abolish, the FSH-induced receptor phosphorylation. The possible impact of phosphorylation on the func- tions of the receptor was examined by testing if conditions that lead to phosphorylation decrease the ability of FSH to stimulate cAMP synthesis. Our data show that as with the addition of FSH, addition of a phorbol ester also results in a decrease in the ability of FSH to stimulate CAMP synthesis.


Follitropin (FSH) and a Phorbol Ester Stimulate the Phosphorylation of the FSH Receptor in Intact Cells*
(Received for publication, August 12, 1993, andin revised form, December 29, 1993) Josh Quintana Using a cell line stably transfected with the rat follitropin (FSH) receptor cDNA we demonstrate that the FSH receptor becomes phosphorylated when cells are exposed to FSH. Since binding of FSH to its receptor results in an increase in CAMP and inositol phosphate accumulation, we examined the potential involvement of protein kinase A and C in mediating receptor phosphorylation. Stimulation of protein kinase A does not appear to be necessary because hFSH-induced receptor phosphorylation was minimally impaired in a cell line that overexpresses cAMP phosphodiesterase. Moreover, stimulation of the protein kinase A pathway with other agonists result in minimal phosphorylation of the FSH receptor. Stimulation of the protein kinase C with a phorbol ester did result in an increase in receptor phosphorylation, and down-regulation of the protein kinase C decreased, but did not abolish, the FSH-induced receptor phosphorylation.
The possible impact of phosphorylation on the functions of the receptor was examined by testing if conditions that lead to phosphorylation decrease the ability of FSH to stimulate cAMP synthesis. Our data show that as with the addition of FSH, addition of a phorbol ester also results in a decrease in the ability of FSH to stimulate CAMP synthesis.
It is now generally accepted that phosphorylation of catecholamine receptors is one of the events involved in the termination of catecholamine actions. A number of studies conducted using the &-adrenergic receptor (reviewed in Refs. [1][2][3][4] gave rise to a model wherein receptor phosphorylation is catalyzed by two distinct classes of serinelthreonine kinases which are activated as a consequence of agonist binding. The agonistoccupied receptor activates Gs which in turn activates adenylyl cyclase resulting in an increase in CAMP synthesis and activation of protein kinase A. At low agonist concentrations, the Pz-adrenergic receptor is phosphorylated by protein kinase A a t serine residues located in one or both of the protein kinase A consensus sites present in the third cytoplasmic loop and in the proximal portion of the C-terminal cytoplasmic tail. This event disrupts the coupling of the receptor to Gs. At high agonist concentrations, the dissociated P-y subunits of Gs recruit the * This work was supported in part by Grant HD28962 from the National Institutes of Health and by funds from the Roy J. Carver Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with  P-adrenergic receptor kinase to the membrane which in turn phosphorylates the agonist-occupied receptor on serine and/or threonine residues located in its extreme C terminus. This P-adrenergic receptor kinase-mediated phosphorylation also disrupts the coupling of the receptor to Gs and promotes the interaction of the receptor with p-arrestin, further uncoupling the receptor from Gs. The model described above is now being tested with other G protein-coupled receptors, but most of these studies are being performed with the members of this family that bind small ligands (5)(6)(7)(8)(9)(10). With regard to the G protein-coupled receptors that bind large ligands, it is known that these receptors also undergo agonist-induced uncoupling (11)(12)(13)(14), but there is little information regarding the role of phosphorylation in this process. Thus, although we have recently initiated studies on the phosphorylation of the lutropidchoriogonadotropin (LWCG)l receptor (15) this phenomenon has not been investigated with the closely related follitropin (FSH) and thyrotropin receptors. Studies on the phosphorylation of these glycoprotein hormone receptors are important for several reasons. First, like the P2adrenergic receptors, the glycoprotein hormone receptors activate the Gsladenylyl cyclaselprotein kinase A pathway, but lack the strong consensus sites for protein kinase A-catalyzed phosphorylation present in the third cytoplasmic loop and the proximal region of the C-terminal cytoplasmic tail of the &-adrenergic receptor (16,17). Weak consensus sequences for protein kinase A-catalyzed phosphorylation are present in some of the intracellular loops and the C-terminal cytoplasmic tail of the glycoprotein hormone receptors, however (16). Second, unlike the &-adrenergic receptor which appears to exclusively activate the Gsladenylyl cyclaselprotein kinase A pathway, the glycoprotein hormone receptors also interact with other G protein(s) leading to increases in inositol phosphates and intracellular calcium (18-21). These events could ultimately result in the protein kinase C-catalyzed phosphorylation of the glycoprotein hormone receptors at one or more of the protein kinase C consensus sequences present in their intracellular regions (16,17). Last, sequence alignments of the three glycoprotein hormone receptors among themselves or with the pzadrenergic receptor, reveals a surprisingly low degree of homology in the intracellular loops and C-terminal cytoplasmic tail, where phosphorylation events are likely to occur (16, 22).
Since our initial studies on the phosphorylation of the LWCG receptor (15) have revealed some potentially interesting differences between it and the &-adrenergic receptor, we initiated a series of experiments designed to study the phosphorylation of the closely related FSH receptor. Our initial studies on the phosphorylation of the FSH receptor are presented here and reveal some interesting similarities as well as differences between it and the LWCG receptor.  cclls wrrr srlrctcd for rrsistancr to hygromycin 11 1250 pghnl) and clonrd as drscrihrd rlsrwherr ( 2 6 ) . Thr chosrn clonal cell linc is drsignatrd 293F(wtl)+P and is mnintninrd in thr mrdium drscrihrd ahovr furthrr supplrmcmtrd with 250 p g h l hygromycin R.

RESULTS
&mist-indtrrrd Phosphoplntion of thr F S N Rrcrptor-In a recent publication, we dcscrihed a polyclonal antihody (Anti-F) that can he used to immunoprecipitate the FSH receptor from metabolically laheled cells (28). Using this experimental approach we showed that the mature FSH receptor expressed hy a cell line (designated 293Ffwtl)) permanently transfected with the rat FSH receptor cDNA is a 74-kDa glycoprotein (28). ;~n d t h a t rocrptor phosphorylation is stimulated hy hFSH. An incrrasr in phosphorylation can he detectrd within 2 min of hormonr :lddition. a maximal increase (ahout 4-fold n v w hnsnl I is attninrtl t)y 15 min and this increase is sustained for at lrast u p to :IO min nftrr hFSH addition. Receptor phosphorylation was not rx:lrninvtl using incuhntions longrr than 30 min. Thew short incuh:ltionr; were chosen to avoid or minimize thc drplrtion of thv s u r f n c r receptors that occurs as a consequenrc of t h r rrcrptor-mc.dintc.d endocytosis of the hormone. Fig. 1 also shotvs that prrimmunr serum does not immunoprecipitatr the phoephorylntrd FYlI receptor from cells incuhated without hFSH o r from rcblls incuhated with hFSH for 30 min. Last, : l l t h o u~h t h v 74-kI)n h:lntl previously identifird as the FSH rrcrptor 12x1 is t h r m w t prominent hand present in thr Anti-F immunoprt,eipit;ltc.s of :'"P-Iahclcd cells. a 171-kDa hand is also prrsrnt. This h:lntl wns detected in some. hut not all, of t h e r x p r r i m r n t s p r r s r n t r d h r r r Phosphorylation of Gonadotropin Rwcptors 171 kD-74 kDaand i t is not detectahlc in immunoprecipitates of [:'5S]cysteinelaheled cells or on Wcstern hlots (28). Thr identity of this protcin(s) has not hecn invcstigatcd.
During a 15-min incuhation an increase in receptor phosphorylation could hc first drtected with ahout 100 ng/ml hFSH, and the maximal rcsponse ( a 4-fold incrrasc in phosphorylation) appear to occur with 1000-2000 ng/ml hFSH (Fig. 2). Thc concentrations of hFSH rcquircd to elicit a dctcctahlc incrcasc in receptor phosphorylation arc rather high compared to those required to rlicit an incrcasc in cAMP synthesis, hut arc comparahle to those rrquircd to incrcasr inositol phosphates (ser helow ).

Phosphorylation of the F S N Rrcrptor t?v Stimulation of Src-
ond Mrssrngrr-actirlat~d Protrin Kinnsrs-In the next scrics of experiments, wc examined thc possihility that activation of the second messcngrr pathways utilized by FSH could result in phosphorylation of the FSH receptor. Sincc it is clear that the LH/CG (15. 18, 19) and the thyrotropin rcccptors (20, 21) mediatc an increase in cAMPand inositol phosphate accumulation in transfectrd cells, wc first examined thr possihilitv that hFSH can also elicit these cffects in 293Fcwtl) cclls.
The effrcts of hFSH on cAMP accumulation shown in Fig. 3 response to hFSH, the degree of phosphorylation of the FSH receptor induced hy hFSH in :32P-lahelcd 293F( w t l )+P cells is similar to that induced in the parrntal :32P-lahcled 293Flwtl) cells. The data presented in Fig. 4 also show that activation of the cAMP signaling systrm with prostaglandin E.,,2 or hy addition of 8-Br-CAMP results in little or no incrrase in the phosphorylation of the FSH receptor in :"P-Iaheled 293F(wtl) crlls. Fig. 4 also shows that addition of a phorhol cstcr t PMA) leads to phosphorylation of thr FSH rrcrptor, whilr addition of a calcium ionophore tA23187) has littlr or no effect. An inactivr phorhol ester (4tu-phorhol 12.13-diacetate) was also inrffcctive (data not shown ). In othrr experiments (not shown ), we examined receptor phosphorylation as a function of time ( A further test for the possihlc involvement of the protein kinase C in thr FSH-inducrd receptor phosphorylation was performed hy assessing receptor phosphorylation in 293Flwtl) cells that had heon preincuhatcd with a high concentration of PMA to "down-regulate" protein kinase C activity (37,38). As shown in Fig. 6 this procedure enhanced basal phosphorylation while eflectivcly down-regulating the prokin kinase C such that freshly added PMA was unahle to increase rrceptor phosphorylation. In contrast, the hFSH-induced recrptor phosphorylation was reduced. hut not abolished. Taken together these data show that addition of hFSH and PMA to ~'2PP-lahclcd 293F(wtl) cells leads to increased phosphorylation of the FSH receptor. Our data also suggest that: ( i ) protein kinase C, but not protein kinase A, is involved in the A t thr concrntrntion usrd, prostaglandin El, stimulatrs cAMP ;ICCIImulntion to a h o u t t h r snmr r x t r n t a s maximally rfTrctivr concrntrntions of hFSH. wrrr thrn addrtl :IS indicatcd. and thr incuhation was continurd for a n additionnl 15 min. Lysatrs were prcparrd and the FSH rrcrptor was immunoprrcipitntrd with Anti-F. Samples wrrr rlrctrophorctically transfrrrrd to polyvinylidrnr difluoridr mrmhrancs. hydrolyzcd. and subjcctrd to thin-layer rlrctrophorrsis as drscrihrd undrr "Exprrimcntal Procrdurrs." Only thr rrlrvnnt portion of a rrprrsrntativr nutoradiogram are shown. Thr dirrction of migration is from hol/om to /op and thr position of migration of authrntic standards is shown. buffer only, or stimulated with hFSH or PMA. Analysis was also performed on 293F(wtl)+P cells incubated with or without hFSH. These results are presented in Fig. 7 and show that phosphoserine and phosphothreonine, hut not phosphotyrosine, can he detected in the FSH receptor phosphorylated in response to hFSH or PMA stimulation.

Functionnl Correlntrs of FSH Rrcrptor Phosphorylation-
The possihle effects of receptor phosphorylation on receptor functions were tested by measuring the ahility of FSH to stimulate cAMP synthesis. This parameter was chosen hecause it is known that preincubation with FSH leads to a subsequent decrease in the ahility of FSH to stimulate adenylyl cyclase (a phenomenon that we call uncoupling, see Refs. 11 and 12) and because agonist-induced phosphorylation of the &-adrenergic receptor is rcsponsihle for a similar phenomenon (reviewed in A correlation hetween hFSH-induced receptor phosphorylation and changes in receptor function is diflicult to test, hecause we do not yet have a paradigm wherein hormone hinding occurs hut the receptor is not phosphorylated. The current expcriments indicate that like receptor phosphorylation Ccf. Fig. 4), the hFSH-induced uncoupling of the FSH receptor from adenylyl cyclase is not impaired in the 293Flwtl)+P cells (data not shown). A correlation hetween PMA-induced phosphor.vlation and changes in receptor function was more directly ascertained in the experiments summarized in Fig. 8. These experiments show that preincuhation of293F(wtl) cells with PMAresults in a subsequent decrease in the ahility of hFSH to increase cAMP synthesis. This effect is noticeahle at all concentrations of hFSH tested, hut its magnitude appears to decrease as the concentration of hFSH increases. We made two additional ohservations that suggest that the functional effects shown in Fig.  8 are largely due to changes in the ability of the FSH receptor to activate cAMP synthesis rather than to changes in the cffector system or to changes in hormone hinding. In order to discern possible changes in the effector system, we examined the effects of PMA on the ability of increasing concentrations of cholera toxin to increase cAMP accumulation. The results (not presented) showed that a preincubation with PMA resulted in a 1.5-2.0-fold enhancement of the effects of cholera toxin on cAMP accumulation. This effect, which is opposite to that shown in Fig. 8, was detected a t all concentrations of cholera toxin tested. Using experimental conditions similar to those used in Fig. 8, we also examined the effects of PMA on the binding on increasing concentrations of ""I-hFSH to the surface (39) of293F(wtl) cells. The results (not presented) showed

Refs. 1-4 ).
t h a t PMA reduces IZ"I-hFSH hinding by ahout 40'; rrgardlrss of the Concentration of hFSH used. kt'hile this rrduction in binding may contribute to the reduced ahilitv of hFSH to increase CAMP accumulation, it is not of suflicirnt magnitudr t o fully explain the drastic reductinn in function dc.tc*ctrtl :It lo\v concentrations of hFSH.
In additional experiments (not presented) we showed that the same experimental protocol used above to down-regulatc the protein kinase C almost completely abolished t h r inhihitory effect of PMA on the FSH-induced CAMP accumulation shown in Fig. 8. These results correlate well with the inahilitv of I'MA to increase the phosphorylation of the FSH rrceptor fnllowing "down-regulation" of the protein kinase C (cf. Fig. 6 ) . Sinw down-regulation of protein kinase C also rrducrd the ahility of hFSH to increase phosphorylation of thc. FSH rcbcrptor ( r f . Fig.  61, we attempted to use the same experimental paradigm to determine if a reduction in the FSH-inclucrd phosphorylation of the FSH receptor resulted in :I reduction in t h r magnitude of FSH-inducrd uncoupling. Howrver. prior to performing thrse experiments, it was necrssary to determine if thr experimrntal approach used in Fig. 8 could h r modified to study the FSHinduced uncoupling. To this end, 293F(wtl) cclls wen' incuhated with hFSH (first incubation) followrd hy rc~moval of t h r free hormone hy extensive washing with a isotonic huffrr of neutral pH, and removal of the rcceptor-hound hormonc, hy a brief exposure of the cells to an isotonic huffrr a t pH 3 (391. A second incuhation of the cells with hFSH was then prrformrd as shown in Fig. 8 and cAMP accumulation was mcvwurrd. In spite of the fact that the acid trratment rrmovc's thc m:ljority of the receptor-bound hormone (as judgrd hv mrasuring thr rolease of '"'I-hFSH) the use of thc rxperimrntal approach usrd in Fig. 8 to study FSH-induced uncoupling \vas prc.clutlrd hecause of the high "hasal" levels of CAMP detrctcd during the second incuhation in the cells that had hren prrincuhatcv! with hFSH. This is prohahly due to the low Ievrls of hFSH that are capahle of increasing cAMP synt.hesis in intact cells (cf. Fig. 31. For rxample, if we use 100-1000 ngiml hFSH in thr prrincuhation usrd to induce uncoupling, :I rrsidual Iwel of hFSH corresponding to only 1-0.1'; ri.r. 1 ngiml hFSH I is (,nough to significantly elevate the hasal Irvc,ls of cAMP ohservrd during the second incuhation ( i . r . the incuhation nerdrd to mc~nsurcuncoupling) hecause the EC,,, for t h r hFSH-induct-d incrtwsr in cAMP accumul:ltion is only 2.9 ng/ml ( c f . Fig. -

31.
Another approach that can he usrd t o study uncoupling is similar to that drscrihrd ahow in which crlls are preincuhntcd with PMA or hFSH followed by extcmsivc washing and rrmovnl of the receptor-hound hFSH. Unlike thc. approach dc.scrihed ahovc, however. the second incuhation is not done hy mcBasuring the effects of hFSH on cAMP nccumulation in intact crlls hut rather by measuring the rffrcts of hFSH on :ldcnylyl cycl:~se activity in cell mcmhranrs (11,151. Lynder thrse conditions "residual hFSH" leftover from the first incuhation is not much of a problem hecause the FCC,,, for thr hFSH stirnulation of adenylyl cyclase activity is ahout 50 ng/ml I 121. A s shown in Fig. 9 (rlpprrpanrl ) we can readily demonstrate FSH-inducrd uncoupling of the FSH-responsivr adrnylyl cyclasr using this approach. The results presented show that prrincuhntion of 293F(wtl) cells with FSH has littlr or no effrct on adrnylyl cyclase activity measurrd under hasal conditions (i.v. in the presence of GTP) or whrn stimulated with NaF, hut the magnitude of the FSH-stimulated adtmvlyl cvclasr activity is rc-duced hy 40-60";. The same rxperimrnt;ll approach docs not allow for t h r measurement, of t h r PMA-inducrd uncoupling. however (Fig. 9, lorcvr pnnrl 1. The basal, as wrll as the Napor FSH-stimulated adcnylyl cyclasr activity of mrmhrantbs prcapared from PMA-treated 293F(wtl) cells is thc. samc. or highrr than that of the control cells. IVr havr puhlishrd similar rrsults  Effect of hFSH and PMAon adenylyl cyclase activity of 293F(wtl) cells. Cells were preincubated for 30 min at 37 "C with the indicated additions. In the top panel the concentration of hFSH used during the preincubation was 100 ng/ml. In the bottom panel, the concentration of PMA used during the preincubation was 200 nM. At the end of this incubation membranes were prepared and assayed for adenylyl cyclase activity (see "Experimental Procedures") in the presence of 100 p~ GTP, 100 p~ GTP, and 1000 ng/ml hFSH, or 10 mM NaF as indicated. Each point shows the average 2 S.E. of six determinations (two experiments with triplicate wells in each).
for the LWCG-sensitive adenylyl cyclase (15). Thus, while the protocol described in Fig.  8 allows for the measurement of PMA-induced uncoupling, it cannot be used to measure FSHinduced uncoupling. Conversely, the protocol described in Fig. 9 allows for the measurement of FSH-induced uncoupling but it cannot be used to study PMA-induced uncoupling. Since we cannot measure the PMA-induced uncoupling using the protocol described in Fig. 9, we cannot use this approach to determine if down-regulation of the protein kinase C reduces the magnitude of the FSH-induced uncoupling because we cannot confirm that the PMA response is lost (or reduced) in the cells with down-regulated protein kinase C.
In summary, although we have been able to document that protein kinase C is partially responsible for the hFSH-induced phosphorylation of the FSH receptor (cf. Fig. 6), we have not been able to determine to what extent this is responsible for the hFSH-induced uncoupling of the FSH receptor from adenylyl cyclase. Since some of the biochemical properties of the PMAinduced uncoupling are different than those of the FSH-induced uncoupling ( c f . Fig. 9) we can safely conclude that the FSH-induced uncoupling cannot be entirely explained by the activation of protein kinase C, however.
Finally, it is also possible that a preincubation with PMA or hFSH also affects the ability of hFSH to increase the levels of inositol phosphates. This possibility is difficult to test because PMA can also have direct effects on the different components of this effector system (ie. the G protein(s) involved, phospholipase C, and/or the enzymes that degrade the inositol phosphates) (40) that cannot be independently ascertained. Thus, while the possible effects of PMA on the Gdadenylyl cyclase effector system could be ascertained by using cholera toxin (see above) we do not know of another ligand (or pharmacologic agent) that could be used to test for these putative effects on the inositol phosphate cascade of 293 cells.

DISCUSSION
There are many biological effects that result from the binding of FSH to its cognate receptor. The results presented here add two novel effects to this list: an increase in inositol phosphate levels, and an increase in the phosphorylation of the FSH receptor.
The data showing that hFSH can increase inositol phosphate levels in cells transfected with the FSH receptor cDNA is consistent with recent observations made with the closely related LWCG (18,19) and the thyrotropin receptors (20,21), and add this receptor to the growing list of G protein-coupled receptors that activate more than one signaling system (see  for additional examples).
From our perspective, the most interesting observation presented here is that the FSH receptor is phosphorylated in intact cells, and that its phosphorylation state can be modulated by stimulation with hFSH o r with a phorbol ester. There are some important conclusions that can be made by comparing the data presented here with those previously published for the &-adrenergic receptor and the LWCG receptor. First, there appears to be a clear difference between the phosphorylation of &-adrenergic and the FSH receptors in response to their respective agonists. In the case of the &-adrenergic receptor, protein kinase A is one of the two kinases involved in agonistinduced receptor phosphorylation ( 1 4 ) while the involvement of this kinase in the phosphorylation of the FSH receptor appears minimal as demonstrated in the studies presented here. The same contrast can be made between the FSH receptor and the closely related LWCG receptor, where experimental approaches similar to those utilized here lead us to conclude that protein kinase A is partially responsible for the hCG-induced phosphorylation of its receptor (15). Another potentially important difference between the phosphorylation of the two gonadotropin receptors is that the FSH receptor is phosphorylated in both serine and threonine residues (Fig. 71, while only phosphoserine can be detected in the phosphorylated LWCG receptor.3 Second, while the p,-adrenergic receptor can be phosphorylated in vitro by protein kinase C, this kinase is not believed to be activated in cells stimulated with a Pz agonist or to be involved in the agonist-induced phosphorylation of the &-adrenergc receptor in intact cells (44). In contrast, activation of the FSH receptor presumably results in the activation of protein kinase C because this receptor has now been shown to generate at least one of the two second messengers (inositol phosphates, see Fig. 3) associated with this signaling system. Moreover, as shown here, stimulation of intact cells with PMA results in phosphorylation of the FSH receptor while downregulation of protein kinase C reduces the magnitude of the hFSH-induced receptor phosphorylation. Thus, it is likely that protein kinase C partly mediates the phosphorylation of the FSH receptor induced by hFSH. In this respect, the FSH receptor behaves in a similar fashion to the LWCG receptor which is also phosphorylated in response to stimulation of cells with PMA (15). The putative roles of protein kinase A and C in the phosphorylation of the rat gonadotropin receptors are consistent with the existence of known consensus sequences for these kinases in the intracellular regions of these two receptors. Both gonadotropin receptors have only 2-3 weak consensus sequences for protein kinase A-catalyzed phosphorylation, but they have 10-12 weak and 1-2 strong consensus sequences of protein kinase C-catalyzed phosphorylation (see Refs. 15 and 16 for a partial list). Last, the results obtained in the cells with down-regulated protein kinase C suggest that, in addition to the protein kinase C, a second messenger-independent kinase(s), such as p-adrenergic receptor kinase, is also involved in the agonist-induced phosphorylation of the FSH receptor.
While the concentrations of hFSH required to increase receptor phosphorylation (ECS0 ? 500 ng/ml) are much higher than those that elicit CAMP accumulation 1 3 ng/ml), they are comparable to those that elicit increases in inositol phosphate accumulation = 100 ng/ml). These data are consistent with our conclusions regarding the lack of involvement of protein kinase A, and the involvement of protein kinase C as well as second messenger-independent kinases in the hFSH-induced receptor phosphorylation (see above). An additional interpretation of these results is that the occupied receptor is a better substrate than the free receptor for the receptor kinase(s). Since the phosphorylation experiments were done using short incubations when hormone binding is not expected to be a t equilibrium, only high hormone concentrations are likely to lead to a high fractional occupancy ofthe receptor population that is presumably needed to detect phosphorylation.
While it is clear that phosphorylation is responsible for the agonist-induced uncoupling of the catecholamine receptors from their respective effectors (1)(2)(3)(4), a cause-effect relationship between agonist-induced phosphorylation and uncoupling of the LWCG and FSH receptors has yet to be established (this paper and Refs. 11,12,and 15). A requirement for ATP during uncoupling of the gonadotropin receptors (12), however, is consistent with the possibility that agonist-induced phosphoryla-R. W. Hipkin, Z. Wang, and M. Ascoli, unpublished observations. tion is involved in the agonist-induced uncoupling of these receptors. Phosphorylation of the gonadotropin receptors induced by their respective ligands or PMA stimulation does correlate with a functional uncoupling of these receptors from at least one of their effector systems ( Fig. 8 and Refs. 12 and 15).
Clearly, more work is needed to fully understand the involvement of protein kinase A and Cs, as well as second messengerindependent kinases (such as a P-adrenergic receptor kinasetype kinase) in the hFSH-induced phosphorylation of the FSH receptor. The location of the phosphorylated serine and threonine residues also needs to be determined. The work presented here, however, sets the foundation for the rational construction of gonadotropin receptor mutants that may not be phosphorylated. Structural and functional analysis of the phosphorylation of these mutant receptors in response to several stimuli will now be used to identify the specific residues that are phosphorylated and to conclusively determine if receptor phosphorylation has an impact on receptor function.