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Endocrinology Vol. 145, No. 8 3594-3602
Copyright © 2004 by The Endocrine Society

Regulation of Gonadotropin-Releasing Hormone Receptors by Protein Kinase C: Inside Out Signalling and Evidence for Multiple Active Conformations

Christopher J. Caunt, James N. Hislop, Eamonn Kelly, Anne-Lise Matharu, Lisa D. Green, Kathleen R. Sedgley, Ann R. Finch and Craig A. McArdle

Laboratories for Integrative Neuroscience and Endocrinology (C.J.C., L.D.G., K.R.S., A.R.F., C.A.M.), and Department of Pharmacology (E.K., A.-L.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; and University of California, San Francisco (J.N.H.), San Francisco, California 94143-2140

Address all correspondence and requests for reprints to: Craig A. McArdle, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol BS1 3NY, United Kingdom. E-mail: craig.mcardle{at}bris.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Desensitization and internalization of G protein-coupled receptors can be mediated by phosphorylation within the C-terminal tail, facilitating ß-arrestin binding and targeting the receptor for internalization. Type II GnRH receptors (GnRH-Rs) show such regulation, but type I GnRH-Rs lack C-tails and are not rapidly desensitized or internalized. Here we show contrasting susceptibility of type I (human and sheep) and II (Xenopus) GnRH-Rs to regulation by protein kinase C (PKC). When human (h) or Xenopus (X) GnRH-Rs were expressed using recombinant adenovirus, PKC activation increased radioligand binding to XGnRH-Rs but not to hGnRH-Rs. A dominant-negative dynamin mutant (K44A) inhibited internalization of XGnRH-Rs (but not hGnRH-Rs) without influencing PKC regulation of XGnRH-R binding. PKC activation increased the affinity of XGnRH-Rs for the type II GnRH ligand and increased effects of low concentrations of GnRH-II on the [Ca2+]i but had no effect on type I ligand binding to hGnRH-Rs, sGnRH-Rs or XGnRH-Rs, or to chimeric receptors with the XGnRH-R C-tail added to a type I receptor. Binding of type II ligand to human or sheep receptors was also unaffected but was increased in the chimeras. Mutation of both PKC-phosphorylation consensus sites in the XGnRH-R tail did not prevent the PKC-mediated increases in binding or alter agonist-induced translocation of ß-arrestin2/green fluorescent protein or inhibition of inositol phosphate accumulation by ß-arrestin2/green fluorescent protein. Thus, it appears that there are two distinct active conformations of XGnRH-Rs (differing in affinity for type I and II ligands) and that these cells exhibit a novel form of inside-out signaling in which PKC feeds back to influence receptor affinity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH (pGLU-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, GnRH-I) acts via G{alpha}q-coupled G protein-coupled receptors (GPCRs) to stimulate phospholipase C (PLC). The consequent mobilization of Ca2+ and activation of protein kinase C (PKC) isozymes mediates secretion of LH and FSH (1, 2, 3). Most vertebrates express the highly conserved GnRH-II ([His5, Trp7, Tyr8]GnRH) along with one or more related peptide. These different forms of GnRH have apparently evolved in parallel with distinct forms of the GnRH receptor (GnRH-R) (3). The best-characterized GnRH-Rs (type I) are selective for GnRH-I. They include all known mammalian GnRH-Rs except for the type II GnRH-Rs recently described in primates and, unlike all other GPCRs, lack C-terminal tails. Type II GnRH-Rs have higher affinity for GnRH-II than for GnRH-I. They have C-terminal tails of varying length and include all known nonmammalian GnRH-Rs (4, 5, 6, 7).

Sustained stimulation causes desensitization and internalization of GPCRs and the established model for rapid homologous GPCR regulation involves their phosphorylation by G protein receptor kinases (GRKs) (8, 9, 10). This occurs in the receptor’s C-terminal tail or third intracellular loop and facilitates binding to ß-arrestins, reducing G protein coupling and targeting the desensitized receptor for internalization via clathrin-coated vesicles. These clathrin-coated vesicles are then pinched off from the plasma membrane by a dynamin collar. This effect requires dynamin’s intrinsic GTPase activity and can be blocked by GTPase inactive (dominant negative) mutants such as K44A dynamin (11). The internalized receptors are then recycled back to the surface membrane or degraded in lysosomes or proteosomes (8, 9, 10). Because internalization of type II GnRH-Rs is slowed by removal of C-tail phosphorylation sites and is accelerated by ß-arrestins, the established model appears applicable to these receptors (12, 13, 14). In contrast, it has not been possible to demonstrate agonist-induced phosphorylation or ß-arrestin binding with type I GnRH-Rs. This apparently reflects the unique absence of C-tails from these receptors and explains their resistance to desensitization and slow rate of internalization (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). As a further distinction, we have expressed hGnRH-Rs and XGnRH-Rs in HeLa cells expressing K44A dynamin and found that the internalization of the type I GnRH-R is insensitive to dynamin, whereas that of the type II receptor is dynamin-dependent (22). Thus, the type I GnRH-R appears atypical, if not unique, in that agonist-stimulated internalization of the nondesensitized GPCR occurs in a phosphorylation, ß-arrestin, and dynamin-independent manner.

In addition to GRKs, many GPCRs are phosphorylated by second messenger regulated kinases such as PKC that can cause heterologous desensitization and influence the rate of internalization and the efficiency of signaling to distinct effector pathways (10). It has long been known that GnRH-Rs mediate PKC activation (23), and early studies revealed no role for PKC in the internalization and down-regulation of endogenous rat pituitary (type I) GnRH-Rs (24). However, the observation that PKC activation caused phosphorylation of a type II (catfish) but not a type I (rat) GnRH-R, raises the possibility that PKC might specifically regulate type II GnRH-Rs (13). Here we have explored this possibility using recombinant adenovirus (Ad) to express GnRH-Rs in HeLa cells conditionally expressing K44A dynamin 1. We show that the type II GnRH-R is, indeed, heterologously regulated (as revealed by a phorbol ester-stimulated increase in radioligand binding) but that this is due to an increase in affinity rather than altered trafficking and is specific for the type II ligand. This increase in affinity is associated with an increase in Ca2+ mobilization by low concentrations of GnRH-II, implying that PKC activation switches the receptor between distinct active receptor conformations, an effect that is reminiscent of the inside-out signaling seen with integrin receptors. We also show that susceptibility to such regulation is conferred by the C-tail but is not prevented by mutation of the two putative PKC phosphorylation sites within the tail. Thus, we have found a novel form of regulation in which ligand binding affinity and specificity are acutely altered by PKC activation and may be mediated by proteins directly or indirectly scaffolded to the receptor via the C-tail.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and cell culture
GnRH and chicken GnRH II (GnRH-II) were purchased from Peninsula Laboratories Europe Ltd. (Mersyside, UK) or from Sigma (Poole, UK). Buserelin and [125I]Buserelin ([T-BuSer6, Pro9 NHET]GnRH-2000 Ci/mmol) were provided by Prof. Sandow (Aventis Pharma GmbH, Frankfurt, Germany). [125I]GnRH-II (~3400 Ci/mmol as determined by self-displacement) was prepared using chloramine-T and purified by G25 Sephadex column chromatography. Culture media, sera, and plasticware were from Invitrogen Life Technologies, Inc. (Paisley, Scotland, UK) or Falcon (Becton Dickinson, Oxford, UK). The two PKC activating phorbol esters, phorbol 12,13 dibutyrate (PDBu) and phorbol 12 myristate, 13 acetate (PMA), the PKC inhibitor, bis-indoylmaleamide I (BIM) and the negative control compound BIM V, were from Calbiochem (Nottingham, UK) and were stored as 10 mM solutions in Me2SO at –20 C. FuGENE 6 was from Roche (Lewes, UK), and Superfect was from QIAGEN (Crawley, UK).

Recombinant Ad expressing the human GnRH-R (Ad hGnRH-R) and XGnRH-R) were generated as previously described (25). Recombinant E1 deleted Ad encoding ß-arrestin2/green fluorescent protein (GFP) and the h.XGnRH-R or s.XGnRH-R C-terminal tail chimeras were produced according to standard techniques (25). The ß-arrestin2/GFP cDNA was kindly provided by Professor J. L. Benovic (Thomas Jefferson University, Philadelphia, PA). GnRH-R chimeras were initially generated by splicing overlap extension PCR products in a final PCR according to protocols outlined in (26). For the h.XGnRH-R chimera, primers 1) 5'-AAG CTG CAG TTT TTC ACA ATG GTG-3' and 2) 5'-ATG AGG GAG TAA AAT ATC CAT AGA TAA GTG GAT C-3' were used to amplify DNA from wild-type (WT) hGnRH-R cDNA template, whereas primers 3) 5'-CTA TGG ATA TTT TAC TCC CTC ATT CAA AGA GG-3' and 4) 5'-CCC GCT CGA GTC AGA AGA CTG ATT GCA TGG T-3' were used to amplify DNA from a WT XGnRH-R cDNA template in a separate reaction. Regions in italic indicate complementary sequences to hGnRH-R DNA, where normal text indicates complementary sequences to XGnRH-R. Underlined regions denote PstI and XhoI restriction sites. The products of these reactions were then used in a splicing PCR using primers 1 and 4 listed above, and the resultant product subcloned back into a corresponding PstI to XhoI digest of WT hGnRH-R in pCR3 vector (Invitrogen Life Technologies). In a similar fashion, the s.XGnRH-R chimera was generated using 1) 5'-AAA CTG CAG TTT TCC ACA GTG GTG-3' and 2) 5'-ATG AGG GAG TGA AAT ATC CAT ATA TAA GTG GAT C-3' PCR primers, using WT sGnRH-R cDNA as template and 3) 5'-TAT GGA TAT TTC ACT CCC TCA TTC AAA GAG G-3' and 4) 5'-CCC GCT CGA GTC AGA AGA CTG ATT GCA TGG-3' primers in a separate PCR using WT XGnRH-R cDNA as template. Regions in italic indicate complementary sequences to sGnRH-R DNA, where normal text indicates complementary sequences to XGnRH-R. Underlined regions denote PstI and XhoI restriction sites. The products of these reactions were then used in a splicing PCR using primers 1 and 4 listed above, and the resultant product subcloned back into a corresponding PstI to XhoI digest of WT sGnRH-R in pCR3 vector. ß-Arrestin2/GFP, h.XGnRH-R, and s.XGnRH-R chimeric receptors were then subcloned into the Ad transfer vector pXCXCMV. The recombinant viruses were then generated by homologous recombination with pJM17 (Microbix Systems Inc., Toronto, Ontario, Canada) in human embryonic kidney 293 cells, grown to high titer and then purified by CsCl density gradient centrifugation as previously described (25). S315A, S332A, and S315A/S332A XGnRH-R PKC consensus-site mutants were generated using the QuikChange PCR-based mutagenesis kit (Stratagene, La Jolla, CA). WT XGnRH-R in a pCR3 vector backbone (Invitrogen) was used as a template in conjunction with 5'-GGA CTT TAC ACT CCC GCA TTC AAA GAG GAC-3' and 5'-GTG AGC ACT CTA CTG GCT AGA AAA GAA AAA AAC-3' mutagenic primers (along with antisense primers), according to manufacturer’s instructions.

Cell culture and transfection
HeLa cells stably expressing K44A dynamin 1 (kindly provided by Dr. S. Schmidt, Scripps Institute, La Jolla, CA) were cultured in serum-supplemented DMEM with G418 (300 µg/ml) and puromycin (100 ng/ml). Tetracycline (1 µg/ml) was routinely included in the culture medium to prevent transgene expression, but in some cases this was omitted to permit K44A dynamin 1 expression and thereby block dynamin-dependent internalization (11). Western blotting was also performed to confirm the effectiveness of this manipulation of K44A dynamin expression as described (22). For experiments, cells were harvested by trypsinization, plated in DMEM supplemented with 2% serum, and incubated for 2 d in flasks or culture plates as described in figure legends. For most experiments, the cells were transfected by infection with recombinant Ad (expressing GnRH-Rs or ß-arrestin2/GFP) as described (22, 25). The Ad-containing medium was removed after approximately 4–6 h and replaced with fresh medium, with or without tetracycline. The cells were then maintained for 1–2 d in culture before use in the binding assays or [3H]IPx accumulation assays. For some experiments, cells were transiently transfected with pCR3 vectors encoding GnRH-Rs with or without ß-arrestin2/GFP, using FuGENE 6 or Superfect and following the manufacturer’s instructions. These cells were also maintained in culture for a further 1–2 d before use in binding assays or [3H]IPx accumulation assays.

Radioligand binding and internalization assays
Radioligand binding to cell surface GnRH-Rs was quantified using whole cell binding assays, either with cells in suspension or with cells grown in culture plates. For suspension binding approximately 50,000 cells were incubated for 30 min at 21 C in 100 µl of physiological salt solution [PSS: 127 mM NaCl, 1.8 mM CaCl2, 5 mM KCl, 2 mM MgCl2, 0.5 mM NaH2PO4, 5 mM NaHCO3, 10 mM glucose, 0.1% BSA, and 10 mM HEPES (pH 7.4)] containing 1 mg/ml bacitracin with approximately 10–10 M [125I]GnRH-II and 0 or 10–10–10–5 M of the unlabeled competitor peptide (22, 25). Free and bound peptide were then separated by centrifugation through oil and radiolabel in the pellet was determined by {gamma}-counting (22, 25). For flat plate binding assays, approximately 50,000 cells (grown in 24-well plates), were washed in PSS and then incubated for 2 h at 4 C in 200 µl PSS containing approximately 10–10 M [125I]GnRH-II and 0 (total binding) or 10–6 M (nonspecific binding) of GnRH-II. The cells were rinsed in ice-cold PSS (three times) and solubilized in 0.3 ml of 0.2 M NaOH with 1% sodium dodecyl sulfate (SDS). Radiolabel in the solubilized cells was determined by {gamma}-counting.

Internalization was determined using a modified flat-plate binding assay in which cells were incubated as above but at 37 C. The cells were rapidly rinsed twice in ice-cold PSS to terminate the incubation and then incubated for 2 min either in ice-cold PSS or in ice-cold 150 mM NaCl, 50 mM acetic acid (pH 3). They were then washed three more times in ice cold PSS and solubilized in 0.3 ml of 0.2 M NaOH with 1% SDS, and this was collected for {gamma}-counting. Specific cell-associated radioactivity was determined by subtraction of nonspecific from the total. Total specific binding is defined as the specific binding in cells receiving no acid wash, whereas acid-resistant (internalized) specific binding is defined as that seen in the acid washed cells. An internalization index was calculated by expressing acid-resistant specific binding as a percentage of total cell-associated specific binding. For most of these assays, the type II-specific ligand, [125I]GnRH-II, was used, and competition was with the unlabeled homologous peptide. For some assays, a type I GnRH-R-specific ligand, [125I]Buserelin was used, again with the homologous unlabeled peptide to define specificity. Details of pretreatments or coincubations with test compounds or control media are given in the figure legends.

Ca2+ imaging and accumulation of [3H]inositol phosphates ([3H]IPx)
The cytoplasmic Ca2+ concentration was measured by video imaging in fura 2-loaded cells as described (27, 28). Briefly, cells were seeded onto glass coverslips at 50,000 cells/ml and infected with Ad XGnRH-R 24 h later. After incubation for a further 24 h, they were washed in PSS and then exposed to 2 µM fura-2/AM (in PSS) for 30 min at 37 C. The coverslips were then washed and loaded into a stainless steel holder fitted into a heating chamber at 37 C. Image capture was performed within 5–25 min of loading in approximately 500 µl PSS, and calibration was as described (28). [3H]IPx accumulation was used as a measure of PLC activity as described (22, 25) using cells labeled by preincubation with [3H]inositol and stimulated in the presence of LiCl. Cells were cultured in 24-well plates in 1 ml of media and 2 µCi [2-3H]inositol (14–16 Ci/mmol) was added to each well for the final 16 h of incubation. After two washes in PSS, each well was stimulated for 30 min with 200–250 µl of PSS containing 10 mM LiCl and appropriate stimuli. These incubations were terminated by adding 1ml of water at 95 C. The cells were lysed by freezing and thawing and [3H]IPx was separated from free [3H]inositol using anion exchange chromatography in formate form Dowex-1 columns. As an internal control for loading, [3H] in IPs was normalized as a percentage of total eluted [3H] as described (22, 25). Details of transfections and stimulation solutions are given in the figure legends.

Confocal microscopy
ß-Arrestin2/GFP redistribution was assessed in human embryonic kidney 293 cells as previously described (29). Briefly, cells were transfected as described above using FuGene (Roche) with 1.2 µg of pCR3 containing XGnRH-R, S315A/S332AXGnRH-R, or empty pCR3 control vector, and 0.3 µg of ß-arrestin2/GFP and grown on coverslips. To assess ß-arrestin2/GFP distribution, cells were incubated for 5min at 37 C control medium or with 1 x 10–6 M GnRH-II or 10–6 M PMA before methanol fixation and mounting in Dako (Carpinteria, CA) fluorescent mount medium. Cells were examined by microscopy on Leica (Deerfield, IL) TCS-NT confocal laser scanning microscope with a Plan-Apo 40 x 1.40 numerical aperture oil immersion objective. All images were collected on Leica TCS-NT software for two- and three-dimensional image analysis and processed in Adobe Photoshop 5.5 (San Jose, CA).

Statistical analysis and data presentation
The figures show the mean ± SEM of data pooled from "n" independent experiments (raw data or data normalized as described in the figure legends) except for the Ca2+ imaging, where "n" indicates the number of cells imaged. Data are typically reported in text as mean ± SEM and statistical analysis was by Student’s t test, accepting P < 0.05 as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
When HeLa cells are infected with recombinant Ad expressing hGnRH-Rs (hGnRH-R) or XGnRH-Rs at a multiplicity of infection of 10 or greater, at least 90% of the cells express GnRH-Rs (22). These receptors are positively coupled to Gq/11, as indicated by stimulation of [3H]IPx accumulation and Ca2+ mobilization. They also have high affinity for receptor-specific ligands [low nanomolar dissociation constants (Kds)] for [125I]Buserelin binding to hGnRH-Rs and for [125I]GnRH-II binding to XGnRH-Rs) and appropriate ligand specificity (rank order of potency Buserelin>GnRH>GnRH-II at hGnRH-Rs and GnRH-II>Buserelin>GnRH at XGnRH-Rs) (22). Possible effects of PKC activators were first explored in K44A HeLa cells cultured in the presence of tetracycline (to suppress K44A dynamin expression) and infected with Ad expressing WT hGnRH-Rs or XGnRH-Rs. After 24 h of culture they were pretreated with a PKC activator (PDBu) or an inhibitor (BIM) before measurement of receptor binding using a whole cell binding assay and radioligands at concentrations below the Kd. As shown (Fig. 1Go), these pretreatments had no measurable effect on binding of [125I]Buserelin to the hGnRH-R, whereas binding of [125I]GnRH-II to the XGnRH-R was increased by PDBu and reduced by BIM. The effect of PDBu was dose dependent (EC50 ~2 x 10–8 M). The effect of PDBu was also mimicked by PMA, a more potent PKC activator (EC50 approximately 10–9 M) (Fig. 2Go), and blocked by BIM but was not influenced by the structurally related negative control compound BIM V (not shown). The effect of PDBu was also mimicked by pretreatment with GnRH-II, although the efficacy of GnRH-II was considerably lower than that of the phorbol ester (Fig. 2Go, inset).



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  		FIG. 1. Effects of PKC activation and inhibition on binding to type I and II GnRH-Rs in HeLa cells. K44A dynamin HeLa cells were cultured with 1 µg/ml tetracycline (to suppress K44A dynamin expression) in 24-well culture plates and infected with Ad expressing hGnRH-Rs or XGnRH-Rs (as indicated). Approximately 24 h later, they were pretreated for 30 min at 37 C with control medium (C), or in medium with 10–6 M PDBu (P) or 10–6 M BIM (B) as indicated. They were then chilled and used in a whole cell binding assay in which they were incubated at 4 C in 150 µl PSS containing approximately 0.2 x 10–9 M [125I]Buserelin (hGnRH-R) or [125I]GnRH-II (XGnRH-R) and the peptidase inhibitor bacitracin (1 mg/ml). After 2 h, the cells were washed (four times) in ice-cold PSS before solubilization in 0.2 M NaOH with 1% SDS and collection for {gamma}-counting. Nonspecific binding was defined by culture in the presence of 10–6 M of the unlabeled homologous competitor peptide. This was typically less than 10% of total binding and was subtracted for calculation of specific binding (shown). The values shown are means ± SEM (n = 3) from a single experiment that is representative of three similar experiments.

 


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  		FIG. 2. Dose dependence of phorbol ester effects on binding to XGnRH-Rs and comparison with GnRH-II. Main panel, Cells were cultured, transfected with XGnRH-Rs, pretreated and used for binding with approximately 0.2 x 10–9 M [125I]GnRH-II as described in Fig. 1Go, except that the pretreatment was for 30 min with the indicated concentration of PMA or PDBu or with control medium (C). Inset, The same protocol was used to compare effects of pretreatments with 10–6 M PDBu and 10–7 M GnRH-II and the vertical axis is the same as main panel. Both panels show mean ± SEM (n = 3) values for specific binding. These are pooled from three separate experiments (each with triplicate determinations) normalized as a percentage of specific binding after pretreatment for 30 min with 10–6 M PDBu. EC50 values were 10–9 M for PMA and 2 x 10–8 M for PDBu.

 
Suspecting that altered cycling during the PDBu pretreatment might influence the availability of GnRH-Rs at the cell surface, we measured receptor internalization in the presence and absence of PMA, and did so in K44A HeLa cells cultured with and without tetracycline to permit or block dynamin-dependent internalization. As shown (Fig. 3Go), the XGnRH-R was rapidly internalized in control cells, and this was largely blocked by expression of K44A dynamin. PMA inhibited XGnRH-R internalization to some extent in control cells but had no such effect when dynamin-dependent internalization was blocked. In contrast, the hGnRH-R was only slowly internalized, and this was not measurably altered by K44A dynamin expression or by PMA (in the presence or absence of K44A dynamin). These data imply that selective inhibition of internalization by PKC could contribute to the receptor-specific increase in binding. However, the increase in XGnRH-Rs binding was unaltered by blockade of the dynamin-dependent (e.g. PKC sensitive) component of internalization (Fig. 4Go). Similar results were obtained in cells expressing WT dynamin under control of the tet-off system. Again, PDBu increased binding in control cells (P < 0.01) and a similar increase was seen in cells expressing the WT dynamin 1 transgene (not shown).



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  		FIG. 3. Effects of PKC activation on dynamin-dependent and dynamin-independent internalization of XGnRH-R and hGnRH-R. Cells were cultured and transfected with XGnRH-R (upper panel) or hGnRH-R (middle panel) as described in Fig. 1Go except that, for the last 18 h of culture they were in medium with 0 or 1 µg/ml tetracycline (to permit or prevent K44A dynamin expression, respectively). They were then incubated for 30 min at 37 C with radioligand and 0 or 10–7 M of the homologous competitor (GnRH-II or Buserelin) before determination of total specific binding and acid-resistant specific binding. The bar graphs show specific acid-resistant binding expressed as a percentage of specific total cell binding (a measure of internalization). The values shown are means ± SEM (n = 3) pooled from three separate experiments (each with triplicate determinations). *, P < 0.05 compared with control without PMA. The lower panel shows a representative Western blot using antibody directed to the HA tag on the K44A dynamin to detect total cellular levels of the transgene in cells cultured with (+) and without (–) 1 µg/ml tetracycline (Tet).

 


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  		FIG. 4. Effect of PKC activation on [125I]GnRH-II binding to XGnRH-R: dynamin dependence. Cells were cultured and transfected with XGnRH-R as described in Fig. 1Go except that, for the last 18 h of culture the cells were in medium with 0 or 1 µg/ml tetracycline (Tet). They were then pretreated with 0 (open bars) or 10–6 M (filled bars) PDBu before being washed and used in a whole-cell binding assay with approximately 0.2 nM [125I]GnRH-II as described in Materials and Methods. The values shown are means ± SEM (n = 3) for specific binding pooled from three separate experiments (each with triplicate determinations) after normalization as a percentage of specific binding in control cells (without tetracycline) after pretreatment with PDBu. Binding was significantly increased by PDBu in control cells (P < 0.01), and a similar increase was seen in cells expressing K44A dynamin.

 
Because a low (below Kd) concentration of [125I]GnRH-II was used in the binding assays above, the increased binding could reflect an increase in affinity and/or an increase in cell surface receptor number. We therefore assessed the effect of pretreatment on binding using low (0.25 nM) or high (12.5 nM) concentrations of radioligand, reasoning that the latter would be sensitive to changes in receptor number but less sensitive to changes in affinity. As shown (Fig. 5Go, upper panel), PDBu pretreatment increased binding with either concentration of radioligand but the effect was more pronounced with the lower concentration. This distinction was evident in a time course study (Fig. 5Go, lower panel) in which PDBu increased the specific binding of 12.5 nM [125I]GnRH-II by approximately 50% but increased the binding of 0.25 nM [125I]GnRH-II to almost 500%. In both cases, the effect was rapid (evident after 5 min), sustained (at least 2 h with the low radioligand concentration) and maximal at approximately 30 min. These data are indicative of an increase in binding affinity and this was confirmed in a competition-binding assay (Fig. 6Go). As expected, [125I]GnRH-II binding to XGnRH-R was inhibited by the homologous competitor peptide and was increased by pretreatment with PDBu. Curve fitting of data from 10 such experiments revealed Kd and maximal binding capacity (Bmax) values of 11.7 x 10–9 M and 148 fmol/well (respectively) in control cells. PDBu pretreatment reduced the Kd to 39 ± 4% of control (P < 0.01) and increased the Bmax to 119 ± 6% of control (P < 0.05). Because these data suggest that XGnRH-R occupancy is increased by pretreatment with PDBu and most evident when low ligand concentrations are used, we tested for functional consequences of this effect at low peptide concentration. As shown (Fig. 6Go, inset), activation of XGnRH-Rs with 10–9 M GnRH-II increased the cytoplasmic Ca2+ concentration from less than 20 x 10–9 M to 602 ± 93 x 10–9 M (n = 20) within 20 sec of stimulation. Although PDBu pretreatment had no measurable effect on the basal Ca2+ concentration, it increased the Ca2+ response elicited by 10–9 M GnRH-II to 1002 ± 37 x 10–9 M (n = 14). Similar amplification was seen in cells stimulated with 10–10 M GnRH-II (not shown) and this effect (like the PDBu-induced increase in binding) was receptor specific because no such increase was seen when cells were infected with Ad sGnRH-R (sheep GnRH-R) and stimulated with 10–9 M GnRH or with 10–10 M Buserelin (not shown). Thus, it appears that PKC activation increases binding affinity (primarily) and thereby increases receptor occupancy and signaling at low ligand concentration.



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  		FIG. 5. Effect of PKC activation on [125I]GnRH-II binding to XGnRH-R: time course and dependence on [125I]GnRH-II concentration. Upper panel, Cells were cultured and transfected with XGnRH-R as described in Fig. 1Go and then pretreated with 0 (open bars) or 10–6 M (filled bars) PDBu before being washed and used in a whole-cell binding assay with 0.25 or 12.5 x 10–9 M [125I]GnRH-II as indicated. The values are means ± SEM (n = 3) for specific binding pooled from three separate experiments (each with triplicate determinations) and expressed in femtomoles/well. Lower panel, Cells were pretreated and binding was assessed using 0.25 or 12.5 x 10–9 M [125I]GnRH-II as above except that the pretreatment period was varied, as indicated. The data shown are pooled from four separate experiments normalized as a percentage of specific binding seen in the appropriate control cells (e.g. the 0 time point for each concentration of [125I]GnRH-II).

 


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  		FIG. 6. Effect of PKC activation on XGnRH-R binding and XGnRH-R mediated Ca2+ elevation. Main panel, K44A HeLa cells were cultured and transfected with XGnRH-R as described in Fig. 1Go, except that they were in T75 flasks. They were then pretreated with 0 ({bullet}) or 10–6 M ({circ}) PDBu for 30 min at 37 C before being collected and used in a 21 C suspension binding assay with approximately 0.2 nM [125I]GnRH-II and competitor, as indicated. The values shown are means ± SEM of duplicate observations in a representative experiment. Curve fitting (GraphPad Prism 2, San Diego, CA) revealed Kd and Bmax values of 11.7 x 10–9 M and 148 fmol/tube (respectively) in control cells. PDBu reduced the Kd to 39 ± 4% of control (P < 0.01) and increased the Bmax to 119 ± 6% of control (P < 0.05). Inset, Ca2+ imaging was performed in K44A HeLa cells cultured, infected with Ad XGnRH-R and loaded with fura/2 as described in Materials and Methods. During imaging, the cells were exposed to 0 (filled symbols) or 10–7 M (open symbols) PDBu for the period indicated by the open bar, and then with 10–9 M GnRH-II (solid bar). The break in the trace represents a period during which images were not captured.

 
To further explore ligand and receptor specificity, we compared effects of PDBu on binding of Buserelin or GnRH-II to type I and type II GnRH-Rs and two chimeric receptors: full-length hGnRH-Rs or sGnRH-Rs with added Xenopus C-terminal tails. These chimeras have pharmacological characteristics (affinity, ligand specificity, and PLC coupling) comparable with WT hGnRH-Rs and sGnRH-Rs in these cells (not shown). Binding of the type-I GnRH-R ligand was lower in Ad hGnRH-R infected cells than in Ad sGnRH-R cells (Fig. 7Go, upper panel). For both of these receptors, addition of the XGnRH-R tail increased binding and because all of these receptors have comparable affinity for [125I]Buserelin (not shown), these differences most likely reflect differences in receptor number. In contrast, the relatively low affinity of XGnRH-Rs for Buserelin explains the minimal binding of [125I]Buserelin to Ad XGnRH-R infected cells. Similarly, binding of [125I]GnRH-II to cells infected with Ad hGnRH-R, sGnRH-R, h.XGnRH-R, or s.XGnRH-R was relatively low because of the low affinity of these receptors for the type II-specific ligand. Despite these differences in expression levels and affinity, it was clear that PDBu pretreatment failed to increase binding of [125I]Buserelin to any of these receptors, whereas it did increase binding of [125I]GnRH-II to each of the receptors with the XGnRH-R C-tail (XGnRH-R, h.XGnRH-R and s.XGnRH-R, Fig. 7Go, lower panel). Thus, the effect is not only receptor specific (seen with cognate ligand binding to type II, but not type I GnRH-Rs) but also ligand specific (seen with [125I]GnRH-II binding to h.XGnRH-Rs and s.XGnRH-Rs, but not when the preferred ligand [125I]Buserelin, binds to these same receptors).



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  		FIG. 7. Effects of PKC activation on binding to GnRH-Rs: ligand specificity and dependence on C-tail. K44A HeLa cells were cultured, transfected and used in whole cell binding assays as described in Fig. 1Go, except that transfection was by infection with Ad expressing WT hGnRH-Rs, sGnRH-Rs, and XGnRH-Rs or chimeric receptors consisting of the entire hGnRH-R or sGnRH-R with the C-terminal tail from the XGnRH-R (h/X and s/X, respectively). Binding was determined for each of these receptors using both of the radioligands and, in each case, nonspecific binding was determined with 10–7 M of the homologous unlabeled ligand. The values shown are means ± SEM (n = 3) for specific binding from a single experiment that is representative of three similar experiments.

 
Because susceptibility to regulation by PKC can be conferred by transfer of the XGnRH C-tail to hGnRH-Rs we considered the possibility that it is mediated by phosphorylation within this structure. To test this receptor, mutants were constructed in which either or both of the putative PKC phosphorylation sites of this region were converted from serine to alanine (S315A, S332A and the double mutant, S315A/S332A, each in pCR3). However, when WT and mutant XGnRH-R were transiently transfected into K44A HeLa cells, pretreatment with PDBu caused comparable increases in binding to each of the receptors (Fig. 8Go).



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  		FIG. 8. Effects of PDBu on binding to XGnRH-Rs: lack of dependence on putative PKC phosphorylation sites within the C-terminal tail. K44A HeLa cells were cultured, transfected with XGnRH-R, pretreated with 0 (open bars) or 10–6 M (filled bars) PDBu and used in binding assays as described in Fig. 1Go, except that the receptors were in pCR3 vector and transfection was by means of the FuGENE 6 reagent. In addition to the WT XGnRH-R, XGnRH-Rs containing one (S315A or S332A) or two (S315A/S332A = double) mutations were used, to remove one or both of the putative PKC phosphorylation sites in the C-tail. The values shown are means ± SEM (n = 3) from an experiment that is representative of five similar experiments. The dual mutation failed to prevent the effect of PDBu pretreatment on binding and similar effects were seen with each of the single mutations (not shown).

 
An alternative possibility is that this regulation reflects phosphorylation (by PKC) of other proteins directly or indirectly scaffolded to the C-tail of these receptors. We therefore also assessed the ability of these receptors to translocate ß-arrestin. When cells were transfected with plasmid encoding ß-arrestin2/GFP, the fluorescent protein was evenly distributed throughout the cytoplasm (Fig. 9Go). Activation of cotransfected XGnRH-Rs caused the ß-arrestin/GFP to translocate to the plasma membrane, indicating agonist-induced receptor phosphorylation and consequent ß-arrestin binding. No such translocation was seen in mock-transfected cells (Fig. 9Go), or in cells stimulated with PMA, irrespective of the cotransfected receptor. Receptor mediated ß-arrestin2/GFP translocation was, however, seen in cells transfected with the double mutant XGnRH-R, implying that this also undergoes ligand-stimulated phosphorylation with consequent ß-arrestin binding. In a final series of experiments, we assessed the functional relevance of ß-arrestin binding to these receptors. In cells transfected with XGnRH-R, GnRH-II stimulated the accumulation of [3H]IPx, and the receptor was rapidly internalized (as measured by a radioligand binding/acid assay). In cells infected with Ad ß-arrestin2/GFP, the GnRH-II-stimulated [3H]IPx response was reduced (from 542 ± 49 to 136 ± 1 cpm/well, Fig. 9Go) and the rate of internalization was increased (from 51.8 ± 5.2 to 79.0 ± 6.9% at 30 min, not shown). In cells transfected with either single or double mutant XGnRH-Rs, the [3H]IPx responses and internalization rates were similar to those of the WT receptor, and these effects were also influenced by Ad ß-arrestin2/GFP in the same way as WT receptors (not shown). Thus, it appears that these two putative PKC phosphorylation sites are not needed for ß-arrestin2 binding or for ß-arrestin2 to influence receptor function.



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  		FIG. 9. Effects of of ß-arrestin2 on receptor mediated [3H]IPx accumulation receptor activation and on ß-arrestin2/GFP translocation. Upper panel, Cells were transfected (FuGENE 6) with cDNA encoding ß-arrestin2/GFP along with the WT or double mutant (S315A/S332A) XGnRH-R or with pCR3 vector alone (mock), as indicated. Distribution of ß-arrestin2/GFP was assessed by confocal microscopy in cells stimulated for 0 (control) or 10 min with 10–7 M GnRH II (GnRH). GnRH II caused a clear translocation of ß-arrestin/GFP in cells expressing either receptor but failed to do so in control (mock transfected) cells just as PMA failed to cause translocation in XGnRH-R expressing cells (not shown). The images shown are each representative of those obtained in at least 10 cells imaged in three similar experiments. Lower panel, Cells were cultured and transfected as described in Fig. 7Go except that, in addition to transfection with the WT or mutant XGnRH-R, they were also infected with control (empty) Ad (open bars) or with Ad expressing ß-arrestin2/GFP (filled bars). For the last 16 h of culture, the cells were labeled with [3H]inositol and [3H]IPx accumulation was then assessed during a 60 min stimulation with 0 (–) or 10–7 M (+) GnRH-II (in the presence of 10 mM LiCl). The values shown are means ± SEM (n = 3) from an experiment that is representative of three similar experiment. ß-arrestin2/GFP inhibited [3H]IPx accumulation mediated via both receptors and had a similar inhibitory effect on each of the single mutant receptors (not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Type I GnRH-Rs do not rapidly desensitize or internalize, characteristics attributed to their unique lack of a C-tail and the consequent absence of agonist-induced phosphorylation and ß-arrestin binding. In contrast, type II GnRH-Rs possess C-tails and, where investigated, show agonist-induced phosphorylation associated with rapid homologous desensitization, internalization, and ß-arrestin binding (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). The type I GnRH-Rs of gonadotropes are coupled via Gq/11 to phospholipase C and PKC, but early studies revealed no role for PKC in receptor internalization or desensitization of GnRH-stimulated gonadotropin secretion (24). This is compatible with the observation that a type I GnRH-R is not phosphorylated in response to agonist or to a PKC-activating phorbol ester, in contrast to the catfish GnRH-R (type II) that was phosphorylated by both stimuli (13). The agonist-induced phosphorylation most likely enables ß-arrestin binding, but the consequences of phosphorylation by PKC are unknown. Here we show that PKC activation in HeLa cells increases binding of [125I]GnRH-II to XGnRH-Rs but has no such effect on binding of [125I]Buserelin to hGnRH-Rs or sGnRH-Rs (Figs. 1Go and 2Go). This provides the first functional evidence for heterologous regulation of these receptors and implies that susceptibility to such regulation may be a further functional distinction between type I and II GnRH-Rs.

PKC activation can alter GPCR internalization (29, 30) raising the possibility that PKC-mediated phosphorylation of type II GnRH-Rs might also influence their trafficking. Because the internalization of XGnRH-Rs and hGnRH-Rs is, respectively, dependent and independent of dynamin in this model, we considered the possibility that PKC-mediated inhibition of dynamin-dependent internalization might selectively increase type II GnRH-R accumulation at the cell surface. Supporting this, we found that XGnRH-R internalization was rapid and dynamin-sensitive compared with the slow and dynamin-insensitive internalization of the hGnRH-R and that PKC activation reduced internalization of the XGnRH-R (but not the hGnRH-R) in control cells, although it had no effect on internalization of either in the presence of K44A dynamin 1 (Fig. 3Go). However, PKC activation caused comparable increases in binding to XGnRH-Rs in control cells and in cells expressing K44A dynamin 1, conditions in which the PKC and dynamin-sensitive internalization is blocked (Fig. 4Go). This uncoupling of PKC effects clearly argues against altered internalization as a cause of the increased binding. In accord with this, competition binding revealed that PDBu pretreatment has little effect on receptor number (Bmax increased approximately 20%) but increases the affinity of XGnRH-R for GnRH-II by approximately 3-fold (Fig. 6Go). Consequently, the effect on binding is more pronounced with a low concentration of radioligand (0.25 nM) than with a high concentration (12.5 nM) (Fig. 5Go). This increase in affinity is functionally relevant because it is associated with an increase in Ca2+ mobilization by low concentrations of GnRH-II (Fig. 6Go). Given the increasing evidence that GPCRs can exist in multiple active conformations (31), the simplest explanation of these data is that GnRH-II binding induces or stabilizes either of two distinct active receptor conformations (which differ in affinity for GnRH-II and are both able to cause a Gq/11 mediated mobilization of Ca2+) and that PKC activation provides a switch that favors use of the higher affinity conformation.

To explore the relevance of the C-tail, cells were infected with Ad hXGnRH-R or sXGnRH-R, and this increased radioligand binding (compared with the WT receptors). Because the chimeras had pharmacological characteristics of other type I GnRH-Rs and there was no corresponding increase in affinity (not shown), this reflects an increase in cell surface receptor number. Similar data were obtained when the catfish GnRH-R C terminus was added to the rat GnRH-R (20, 32). The catfish GnRH-R has two cystein residues in the C-terminal tail, providing the potential for palmitoylation (13), and truncations removing these sites reduce expression, implying that palmitoylation within the tail increases expression by increasing membrane anchorage and stabilization (13). It is therefore of interest that a similar increase is seen with the XGnRH-R tail (Fig. 6Go), although it lacks any palmitoylation site (33). This may reflect the presence of an eighth intracellular {alpha}-helix in the proximal C-terminal tail, as seen in the crystal structure of rhodopsin and postulated for ß2 adrenergic receptors (34, 35). For the XGnRH-R, any such proximal {alpha}-helix would be amphipathic with the potential for receptor stabilization by apposition of a hydrophobic face (consisting of Phe316, Leu320, Trp323, Ile324, Val327, Leu330, and Leu331) with the plasma membrane.

As well as increasing receptor number, addition of the C-tail of the XGnRH-R to type I GnRH-Rs enabled PKC activation to acutely increase binding to GnRH-II, a finding that was unexpected because C-tails are typically involved in mediation and termination of GPCR signaling, rather than alteration of the ligand binding pocket. This is reminiscent of the inside-out signaling seen with integrin receptors. In platelets, for example, the affinity of integrin receptors for their extracellular ligands (e.g. fibrinogen) is rapidly increased by GPCR activation. Such affinity modulation can be mediated by PKC and dependent upon C-terminal domains of the receptors (36, 37, 38). A further intriguing feature of the GnRH-R regulation reported here is that it is ligand specific. The XGnRH-R C-tail enables PKC activation to alter the ligand-binding pocket to increase both affinity and specificity for GnRH-II. Such regulation requires different sites of receptor interaction for the type I and II ligands as indicated by other comparative studies (39). The Trp7 in GnRH-II (Leu7 in GnRH-I) is favored by catfish and chicken GnRH-Rs, but not by type I GnRH-Rs (40), so an increase in affinity and specificity would be anticipated by conformational changes favoring this interaction in the type II receptor and ligand. However, the amino acids in the XGnRH-R interacting with Trp7 are unknown, and it is not clear how such alteration would map onto the type I GnRH-Rs (where type II ligand binding is also increased in the PDBu pretreated chimera).

Although susceptibility to regulation by PKC can be conferred on hGnRH-Rs or sGnRH-Rs by addition of the C-tail, this does not reflect direct phosphorylation of the C-tail because removal of its two putative PKC phosphorylation sites did not prevent regulation by PKC. An alternative possibility is that the pertinent PKC substrates are bound directly or indirectly to the C-tail. It has recently been shown that removal of putative caseine kinase II phosphorylation sites from the C-tail of the TRH receptor prevents ß-arrestin binding, just as inclusion of such sites into the C-tail of the catfish GnRH-R facilitates binding (41). Because the two PKC sites that we have mutated in the XGnRH-R C-tail are also potential caseine kinase II sites, we considered the possibility that they might also influence ß-arrestin binding (41). In exploring this, we found that mutation of these sites in the XGnRH-R did not prevent ß-arrestin2 binding or function (Fig. 9Go). Accordingly, the regulation of XGnRH-R binding does not reflect phosphorylation of the receptor’s C-tail by PKC but could be mediated by phosphorylation of proteins scaffolded to the receptor by C-tail bound ß-arrestin. Although such scaffolding has not been extensively explored for GnRH-Rs, ß-arrestins are known to anchor signaling proteins (such as Src, Jnk, and ERK) in the vicinity of other GPCRs (8, 9, 10).

Because the roles of type II GnRH-Rs are unclear (5), the physiological relevance of their regulation by PKC is unknown. The increase in affinity would be expected to enhance signaling at low receptor occupancy, but physiological levels of type II GnRH-R occupancy are unknown. It has recently been shown that sheep gonadotropes express both type I and type II GnRH-Rs (4), so it is possible that heterologous regulation by PKC favors the activation of the type II GnRH-Rs by type II ligand, as opposed to activation by cross-reacting type I ligand in these cells. Moreover, because responses mediated by type I and II GnRH-Rs may differ (4), such regulation could influence the range of responses in cells exposed to both peptides. Because GnRH-Rs mediate activation of PKC, a further intriguing possibility is that a PKC mediated increase in receptor affinity provides a positive feedback loop for XGnRH-R signaling. In support of this, we have found that pretreatment with GnRH-II causes a rapid increase cell surface receptor binding (Fig. 2Go) and that this effect is inhibited by BIM (not shown). These observations also raise the possibility of cross-talk in which activation of other Gq/11-coupled GPCRs amplifies signaling via type II (but not type I) GnRH-Rs.

In summary, we have described a novel form of GnRH-R regulation in which PKC activation increases the affinity of a type II GnRH-R for a type II ligand. This effect is not only receptor specific but also ligand specific and implies that there are at least two distinct active conformations of the XGnRH-R differing in affinity and ligand specificity. Moreover, the increase in binding is dependent upon transferable sequences within the C-terminal tail of the XGnRH-R but does not reflect direct phosphorylation of its putative PKC phosphorylation sites and may therefore be mediated by other proteins scaffolded to this structure.


   Acknowledgments
 
The K44A Hela cells and the plasmids encoding GnRH-Rs were kindly provided by Profs. S. Schmidt (Scripps Institute, La Jolla, CA) and R. Millar (Medical Research Council Human Reproductive Sciences Unit, Edinburgh, Scotland, UK), respectively.


   Footnotes
 
This work was supported by the Wellcome Trust (062918).

Abbreviations: Ad, Adenovirus; BIM, bis-indoylmaleamide; Bmax, maximal binding capacity; GFP, green fluorescent protein; GnRH-R, GnRH receptor; GPCR, G protein-coupled receptor; h, human; IP, inositol phosphate; GRK, G protein receptor kinase; kd, dissociation constant; PDBu, phorbol 12,13 butyrate; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12 myristate 13 acetate; PSS, physiological salt solution; s, sheep; SDS, sodium dodecyl sulfate; WT, wild-type; X, Xenopus.

Received January 27, 2004.

Accepted for publication March 22, 2004.


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Microbiology, October 1, 2007; 153(10): 3517 - 3526.
[Abstract] [Full Text] [PDF]

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E. L. Baldwin, I. N. Wegorzewska, M. Flora, and T. J. Wu
Regulation of Type II Luteinizing Hormone-Releasing Hormone (LHRH-II) Gene Expression by the Processed Peptide of LHRH-I, LHRH-(1-5) in Endometrial Cells
Experimental Biology and Medicine, January 1, 2007; 232(1): 146 - 155.
[Abstract] [Full Text] [PDF]