Transient Phosphorylation of the V1a Vasopressin Receptor

The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells. The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells. Agonist binding and activation of many G-protein-coupled receptors is followed by desensitization, an attenuation of the cellular response to the ligand that prevents sustained stimulation. Studies with rhodopsin and the β2-adrenergic receptor have implicated phosphorylation in receptor desensitization (1Bennett N. Sitaramayya A. Biochemistry. 1988; 27: 1710-1715Crossref PubMed Scopus (130) Google Scholar, 2Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar) Receptor phosphorylation promotes the binding of arrestin, which in turn uncouples the receptor from the G-protein and enhances sequestration. In the particular case of the β2-adrenergic receptor, it has been postulated that sequestration of the phosphorylated receptor facilitates cleavage of the phosphate groups by phosphatases that associate with the endosomes and allows the recycling of the de-phosphorylated active protein to the cell surface (3Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Data obtained with other G-protein-coupled receptors have indicated that phosphorylation is not required to observe receptor sequestration and desensitization. For example, a truncated form of the V2 vasopressin receptor lacking the phosphorylation acceptor sites at the carboxyl-terminal end was sequestered and desensitized, although to a lesser extent than the wild type receptor (4Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Crossref PubMed Scopus (68) Google Scholar). Similarly, a mutant angiotensin II receptor missing a portion of the carboxyl terminus did not exhibit ligand-induced sequestration although it contained a putative GRK 1The abbreviations used are: GRKs, G-protein-coupled receptor kinases; V1aR, V1a arginine vasopressin receptor; AVP, arginine vasopressin; PMA, phorbol 12-myristate 13-acetate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; D-PBS, Dulbecco's PBS; HBSS, Hanks' buffered saline solution; IP, inositol phosphates; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C; PLC, phospholipase C; TyrPhaa, phenylacetyl-d-Tyr(Et)-Phe-Val-Asn-Lys-Pro-Tyr-NH2; r, receptor; CCK, cholecystokinin. phosphorylation site (5Thomas W.G. Baker K.M. Motel T.J. Thekkumkara T.J. J. Biol. Chem. 1995; 270: 22153-22159Crossref PubMed Scopus (110) Google Scholar). Different kinds of protein kinases can be responsible for receptor phosphorylation. Those that phosphorylate the receptor occupied by agonist are termed G-protein-coupled receptor kinases (GRKs), of which six have been identified up to now (2Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar). With the exception of GRK1 and GRK4, found in the retina and in testis, respectively, the other enzymes are present in most tissues and seem to catalyze in an agonist-dependent manner the phosphorylation of all the receptors of this family that have been tested until now. Receptors can also be phosphorylated by other kinases such as the cAMP-dependent and the Ca2+-dependent (PKC) protein kinases. These enzymes can be activated by an increase in second messengers and may contribute to reduce receptor activity. Some receptors can be substrates to multiple kinases, while others, exemplified by the V2 vasopressin receptor, serve as substrates to a single type of kinases, the GRKs (4Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Crossref PubMed Scopus (68) Google Scholar). Three vasopressin receptor subtypes are known to date: V1a, V1b, and V2 (6Jard S. Kidney Int. 1988; 26: 38-42Google Scholar). Whereas the V2 receptor stimulates adenylyl cyclase activity, the V1a and V1b receptor subtypes activate phospholipases A2, C, and D. Previous studies in hepatocytes and in smooth muscle vascular cells have described ligand-induced desensitization and sequestration of the V1a vasopressin receptor (V1aR), as well as its recycling to the cell surface following removal of the ligand (7Fishman J.B. Dickey B.F. Bucher N.L. Fine R.E. J. Biol. Chem. 1985; 260: 12641-12646Abstract Full Text PDF PubMed Google Scholar, 8Briner V.A. Williams B. Tsai P. Schrier R.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2854-2858Crossref PubMed Scopus (23) Google Scholar). However, data regarding V1aR phosphorylation and its relationship to receptor desensitization are lacking. The availability of the V1aR cDNA (9Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (463) Google Scholar, 10Innamorati G. Lolait S.J. Birnbaumer M. Biochem. J. 1996; 314: 710-711Crossref PubMed Scopus (8) Google Scholar) has enabled the examination of whether the V1aR is phosphorylated and the characteristics of this process. Dulbecco's modified Eagle's medium (DMEM), minimum essential medium without sodium phosphate, Dulbecco's PBS (D-PBS), penicillin/streptomycin, Geneticin (G418), 0.5% trypsin, 5 mm EDTA, and fetal bovine serum were from Life Technologies, Inc.; methionine/cysteine-free DMEM was from ICN, Costa Mesa, CA; cell culture plasticware was from Costar, Cambridge, MA. [3H]Arginine vasopressin (specific activity 60–80 Ci/mmol), Expre35S35S protein labeling mix (specific activity >1000 Ci/mmol), and [32P]H3PO4 in water (pH 5–7) were purchased from NEN Life Science Products. Amplify® was from Amersham Corp. Okadaic acid was from RBI (Natick, MA). Phenylacetyl-d-Tyr(Et)-Phe-Val-Asn-Lys-Pro-Tyr-NH2(TyrPhaa) was from Bachem Bioscience (King of Prussia, PA); AVP, staurosporine and all other reagents were from Sigma. The hemagglutinin epitope (YPYDVPDYA) was introduced using a polymerase chain reaction-based approach (11Birnbaumer M.S. Gilbert S. Rosenthal W. Mol. Endocrinol. 1994; 8: 886-894Crossref PubMed Google Scholar). The resulting construct was sequenced fully by the dideoxy chain termination method of Sanger et al. (12Sanger F. Nicklen S. Coulson A.B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52766) Google Scholar). For expression in eucaryotic cells, the cDNAs were cloned into the expression vector pcDNA3 (Invitrogen, Boston, MA). HEK 293 cells were grown in DMEM-high glucose, supplemented with 10% heat-inactivated fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 μg/ml). Cells were plated at a density of 2.8 × 106 cells/100-mm dish and transfected the following day by a modification of the method of Luthman and Magnusson (13Luthman H. Magnusson G. Nucleic Acids Res. 1983; 11: 1295-1308Crossref PubMed Scopus (464) Google Scholar). Growth medium was replaced with 6.7 ml of a mixture of 100 μm chloroquine and 0.25 mg/ml DEAE-dextran in DMEM with 10% fetal bovine serum containing 3 μg of DNA. After 2 h at 37 the was and the cells were for at with 10% in The cells were with PBS and to medium at 37 were h after Cells were transfected by the phosphate of and PubMed Scopus Google Scholar) as described The day × 106 cells were plated into of The phosphate containing μg of was all reagents were at 37 The reagents were in a in the following of in mm EDTA, mm to the to ml of mm 100 of mm mm 5 mm mm to with reagents were with after After at of the was to containing cells and by After h in the the medium was and cells were with 2 ml of in at 37 After the mixture was with ml of HBSS, with continuous The was and the cells were with medium was and the were to the The day the cells were with the medium containing and into using a were after and in to for binding as described Cells were plated at a density of × in for h with mg/ml were the following Cells were with after which well ml of containing bovine serum and the of were for 2 h on of in the removal of the binding mixture by After with D-PBS, ml of was to well to After at 37 the from the was to containing ml of for binding was the in the presence of μm AVP (11Birnbaumer M.S. Gilbert S. Rosenthal W. Mol. Endocrinol. 1994; 8: 886-894Crossref PubMed Google Scholar). were at of inositol phosphates was by a modification of the method of et al. J. Biol. 1992; PubMed Scopus Google Scholar). transfected cells were grown in culture in medium supplemented with 2 cells were at with ml of supplemented with mm glucose, mm and mm After the cells were at 37 for in ml of the of were to The was for after which ml of the containing the ligand was to the The was for the indicated at 37 the end of the the was rapidly by the and the were by a mixture of and followed by the addition of ml of mm acid to phosphates were from to a described in J. Biol. 1992; PubMed Scopus Google after h on the mm acid were to that been with 2 and mm to with after the were 3 ml of were to and the were in containing ml of was to this of the was in this The were than with ml of mm and were with 5 ml of into containing ml of The accumulation of was by the for by the of the for were in the as by et al. Gilbert S. Birnbaumer M. Birnbaumer Mol. 1994; Google Scholar). Cells were to 100 AVP at 37 in minimum essential medium without sodium phosphate for the phosphorylation and in DMEM-high for all the other to the binding cells were with PBS, for with an containing mm 5 followed by with PBS to removal of the ligand and of the transfected cells were plated in × 106 The following the cells were and for with essential followed by the addition of 100 After h at 37 the cells were to vasopressin other for the and described in the Cells were with phosphate-buffered and for h at in of mm mm 5 mm EDTA, sodium sodium with mm and mm sodium mm was by with an at an of Three were at of were by the addition of of a of protein in for and protein was by for h with bovine serum in followed by in The were at with and the were by with protein for 2 h at The were and 5 with of after by The were from the for at with of containing 10% The were in 10% and by the to of the incorporated into was using a containing the of were to the to be the were expressed as of the phosphorylation obtained by for with 100 was with The day after the cells in were for h in methionine/cysteine-free DMEM glucose, followed by 2 h labeling in the medium containing 100 of Expre35S35S Protein Cells were and the receptor was and as described The were by the with Amplify® followed by of the The obtained with the the binding and the accumulation data were by the method using The of the was with the in The of and were obtained from the transfected cells the V1aR were by AVP labeling with of of the followed by and of the receptor with the as described the of the of the V1aR protein in in a single and (V1aR) was by in from transfected HEK 293 cells. The was not present in HEK 293 cells The V1aR has a of and sites for (9Morel A. O'Carroll A.M. Brownstein M.J. Lolait S.J. Nature. 1992; 356: 523-526Crossref PubMed Scopus (463) Google the of the receptor was with the presence of whether addition of the epitope receptor binding were on cells. The ligand binding obtained was 2 to the by et al. (7Fishman J.B. Dickey B.F. Bucher N.L. Fine R.E. J. Biol. Chem. 1985; 260: 12641-12646Abstract Full Text PDF PubMed Google Scholar) for the V1aR present in The for was at and the of inositol phosphate accumulation was to for the V1aR present in cells S. G. 8: Scopus Google Scholar). whether activation by ligand in receptor cells the V1aR were with to and to hormone. were at following addition of 100 AVP, and the receptor was and by and the time of phosphorylation of the significant amount of was found incorporated into the receptor to the addition of this for as as of the phosphorylation observed after a with AVP, and it be to as phosphorylation. a very increase in at the time the receptor rapidly despite the continuous presence of The of was After the amount of incorporated into the receptor to of the cells with acid to the addition of AVP did not the of the receptor not by the in 2 the extent of V1aR phosphorylation was on the amount of AVP The of phosphorylation on concentration was after of the cells to of phosphorylation not the amount in the V1aR, it also the of the receptor protein in as with the receptor protein with as in of 100 AVP promoted the of 80% of the binding sites from the cell surface with a as in is to been in hepatocytes (7Fishman J.B. Dickey B.F. Bucher N.L. Fine R.E. J. Biol. Chem. 1985; 260: 12641-12646Abstract Full Text PDF PubMed Google and it a phosphorylation and ligand-induced of the The V1aR sites for PKC and receptor signaling this by and the it was to the involvement of PKC in the phosphorylation of the receptor promoted by of cells with 100 for to PKC activity, the of receptor phosphorylation that PKC could phosphorylate the unoccupied receptor. After of the phosphorylation obtained was of the response by AVP, and it could be by the cells with μm of the PKC in following the addition of was a increase in receptor phosphorylation that to those obtained with AVP after addition of the was followed by a of the incorporated into the of the cells with the also in the transient phosphorylation of the V1aR by PKC. of unoccupied V1aR by did not in a of binding sites from the cell surface at of the as in B. PKC phosphorylation by was not sufficient to promote receptor sequestration. of the cells with 100 μm did not promote of into the V1aR a for protein kinase in receptor phosphorylation not The of PKC in V1aR phosphorylation were also in of cells with μm staurosporine by the of into the receptor without the of phosphorylation. The of the cells with μm to PKC activity are in After the phosphorylation of the receptor was of the increase the in cells not with PMA, but it remained The fact that of PKC by staurosporine by with did not of phosphate from the V1aR that phosphorylation of the sites modified by PKC did not a in the of the phosphate groups incorporated into the receptor protein by of PKC on V1aR phosphorylation. Cells the V1aR were used for this After with μm to PKC, and cells were with and to 100 AVP for the The receptor was by as described is of cells not to AVP are identified as C the of GRKs in the mutant was with the V1aR into HEK 293 cells S.J. Mol. 1997; 51: PubMed Scopus Google Scholar). receptor phosphorylation was in the presence and of staurosporine to PKC activity. Although a was in all the not the involvement of GRKs in V1aR phosphorylation. These were the in transfected all other were with transfected cell of the cells with 100 AVP promoted the of than of the binding sites from the a significant in the of the to the of inositol phosphates was to the in receptor in examination of the time of accumulation that the rate of of inositol phosphates remained after the addition of The of with receptor sequestration was but was not The rate of accumulation was at for of desensitization. in the in the rate of accumulation of was in the and to a rate of sustained activity. The sustained activity observed could be to the the receptors present on the cell to sustained activity of the the of the V1aR antagonist in the accumulation of was tested M. A. J. Google Scholar). after addition of 100 AVP, the was and replaced by μm and the of the antagonist on PLC activity was at in addition of the antagonist rapidly the accumulation of that it was mediated by receptors present on the cell surface. of transfected cells with to PKC activity to the and of accumulation of inositol suggesting that was involvement of PKC in this desensitization not The of recycling of the receptor to the cell surface was also After removal of AVP, recycling of receptor to the cell surface was a that been described for the V1aR in smooth muscle vascular cells V.A. Williams B. Tsai P. Schrier R.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2854-2858Crossref PubMed Scopus (23) Google Scholar). in the receptor a AVP rapidly to the cell surface following removal of AVP with an acid and of the cells to the time the of binding sites on the cell surface to very to those present in the cells that a of the receptors been after of the V1aR by agonist promoted phosphorylation of the receptor from been observed with most receptors of this the phosphorylation very and was transient in the continuous presence of was an in the HEK 293 cell the V2 vasopressin receptor was found to phosphorylation at a rate and to the phosphate incorporated into the protein for as as in the presence of phosphorylation in the continuous presence of ligand has been for other receptors as the and the receptors expressed in D. N. P. A. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. M.J. W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google and the receptor to cells J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the and the receptors about 5 after addition of ligand and a in the phosphate of the receptor in cells after addition of the ligand and to after However, the receptor was expressed in cells, it remained phosphorylated at its in the continuous presence of F. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). with the V1aR was the in phosphorylation. The data indicated that protein kinase C and other kinases, most GRKs, were in the The that phosphorylation of the V1aR by PKC the observed was not by with other G-protein-coupled For the R.W. J. A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google angiotensin M. N.J. R.W. Lefkowitz R.J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google and A. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) receptors not phosphate groups in the continuous presence of the although PKC to phosphorylation. is with the that of PKC by inhibition of its activity by staurosporine a significant on the of phosphorylation and of the The longer phosphorylated of the V2 vasopressin and other receptors expressed in HEK 293 cells indicated that transient phosphorylation is not on the cells, rather it is an intrinsic of V1aR of the V1aR phosphorylation in the presence of staurosporine by the that the protein is a for is in to the of GRKs to phosphorylate other receptors of this The of the that phosphorylate this receptor the six known of the have been in HEK 293 cells. whether the V1aR of hepatocytes and smooth muscle vascular cells transient it be to with for the receptor In addition to AVP promoted of the Although the a phosphorylation and of the V1aR, has the of the V2 vasopressin receptor (4Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Crossref PubMed Scopus (68) Google Scholar). that GRK phosphorylation not be for the of the ligand occupied receptor, but to this of the sites phosphorylated by in the V1aR followed by the of the of this mutant V1aR in of the recycling of the receptor to the cell surface. The receptor on the and the of sites to those present at the of the suggesting that recycling without significant of V1aR The of the de-phosphorylated receptor protein to the cell surface be in with the of et al. (3Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar) that as a for the receptor to be de-phosphorylated and its activity, to to PLC activation very rapidly after followed by a in the rate of accumulation of inositol The attenuation of PLC activity was not as as from the significant of receptors from the cell surface. that the rate of accumulation was sustained for than in the presence of while after 5 than of the receptor remained the of the V1aR within the 2 after addition of as in the of and was of of PLC activity at with has been described for in that are until the activity of the J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). The sustained of PLC activity that the desensitization of the rate of accumulation was to the sequestration of the phosphorylated receptor resulting in a in the of PLC stimulation. The of a V1aR antagonist to the sustained accumulation of that followed indicated that the activity of PLC was by receptors present on the cell was by recycling of the with the that a by ligand binding is required to receptor (4Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Crossref PubMed Scopus (68) Google phosphorylation of unoccupied receptor was not sufficient to a of cell surface These data are with the that a truncated V1aR missing the putative PKC sites on the carboxyl terminus ligand-induced desensitization, by ligand-induced of receptor from the cell surface. on V1aR phosphorylation was N. B. A. 1997; PubMed Scopus Google Scholar). Some of the phosphate incorporated in response to was the that could without the receptor to the The phosphate incorporated by PKC into the receptor was at a rate than the phosphate incorporated into the receptor and are by the that of the V1aR the receptor to that are in phosphatases responsible for the that the phosphorylated occupied receptor a that it a for the phosphatases of its The could not a the of the incorporated phosphate and the of the receptor. amount of phosphate was present in the receptor. The in by the of phosphate promoted by receptor activation that are as well as the phosphorylation is to has been described for the β2-adrenergic receptor for which multiple cAMP-dependent protein kinase and GRK phosphorylation sites have been demonstrated to in desensitization and sequestration (2Freedman N.J. Lefkowitz R.J. Recent Prog. Horm. Res. 1996; 51: 319-351PubMed Google Scholar). of phosphorylation were for the V1aR in the In activation of the V1aR by AVP promotes phosphorylation of the receptor by GRK and PKC. PKC phosphorylation by not promote V1aR sequestration its desensitization. phosphorylation is and in the continuous presence of a that intrinsic to the receptor and sequestration of the ligand occupied receptor are and seemed to in desensitization.