Identification of the Sites of N-Linked Glycosylation on the Human Calcium Receptor and Assessment of Their Role in Cell Surface Expression and Signal Transduction
1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês
10.1074/jbc.273.51.34558
ISSN1083-351X
AutoresKausik K. Ray, Peter Clapp, Paul K. Goldsmith, Allen M. Spiegel,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoThe human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site-directed mutagenesis. Asparagine residues of the consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild typeversus mutant receptors demonstrate that, of the 11 potential sites for N-linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, -488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50–90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction. The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site-directed mutagenesis. Asparagine residues of the consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild typeversus mutant receptors demonstrate that, of the 11 potential sites for N-linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, -488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50–90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction. calcium receptor human calcium receptor G-protein-coupled receptor phosphoinositide extracellular domain human embryonic kidney-293 cells Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline polyacrylamide gel electrophoresis peptideN-glycosidase F endo-β-N-acetylglucosaminidase H bovine serum albumin 1,4-piperazinediethanesulfonic acid d-biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester wild type. The calcium receptor (CaR)1 is a G-protein-coupled-receptor (GPCR) involved in extracellular calcium homeostasis by controlling the rate of parathyroid hormone secretion from the parathyroid gland and the rate of calcium reabsorbtion by the kidney (1Brown E.M. Pollak M. Hebert S.C. Annu. Rev. Med. 1998; 49: 15-29Crossref PubMed Scopus (190) Google Scholar). Recent evidence suggests that the CaR is also involved in diverse cellular responses to extracellular calcium within microenvironments in other organs such as brain, skin, bone, and intestine (2Chattopadhyay N. Vassilev P.M. Brown E.M. Biol. Chem. 1997; 378: 759-768PubMed Google Scholar). Extracellular calcium ion ([Ca2+]o) activates the CaR, leading to activation of phospholipase-Cβ via the Gqsubfamily of G-proteins; this increases phosphoinositide (PI) hydrolysis, which in turn causes release of calcium from intracellular stores (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar).The CaR, metabotropic glutamate receptors (4Nakanishi S. Science. 1992; 258: 597-603Crossref PubMed Scopus (2284) Google Scholar), and a subgroup of putative pheromone receptors in the vomeronasal organ (5Herrada G. Dulac C. Cell. 1997; 90: 763-773Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 6Matsunami H. Buck L.B. Cell. 1997; 90: 775-784Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar, 7Ryba N.J. Tirindelli R. Neuron. 1997; 19: 371-379Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar) comprise a unique subset of the superfamily of GPCRs characterized by a relatively large (500–600-residue) amino-terminal extracellular domain (ECD). The ECD of the human CaR (hCaR) (8Garrett J.E. Capuano I.V. Hammerland L.G. Hung B.C.P. Brown E.M. Hebert S.C. Nemeth E.F. Fuller F. J Biol. Chem. 1995; 270: 12919-12925Crossref PubMed Scopus (455) Google Scholar) contains 11 potentialN-linked glycosylation sites (9Shakin-Eshleman S.H. Spitalnik S.L. Kasturi L. J. Biol. Chem. 1996; 271: 6363-6366Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), of which nine are highly conserved in bovine (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar), rat (10Riccardi D. Park J. Lee W.S. Gamba G. Brown E.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (434) Google Scholar, 11Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (343) Google Scholar), rabbit (12Butters Jr., R.R. Chattopadhyay N. Nielsen P. Smith C.P. Mithal A. Kifor O. Bai M. Quinn S. Goldsmith P. Hurwitz S. Krapcho K. Busby J. Brown E.M. J Bone Miner. Res. 1997; 12: 568-579Crossref PubMed Scopus (63) Google Scholar), and chicken (13Diaz R. Hurwitz S. Chattopadhyay N. Pines M. Yang Y. Kifor O. Einat M.S. Butters R. Hebert S.C. Brown E.M. Am. J. Physiol. 1997; 273: R1008-R1016Crossref PubMed Google Scholar) CaRs. The remaining two sites are conserved among human, rabbit, and chicken CaRs, but only one of the two (Asn-386 or -400 in the hCaR sequence) is conserved in rat and bovine CaRs, respectively.The carbohydrate moieties of glycoproteins in general are believed to be important for facilitating protein folding, protection from proteolysis, intracellular trafficking, secretion, and cell surface expression (14Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (558) Google Scholar, 15Opdenakker G. Rudd P.M. Ponting C.P. Dwek R.A. FASEB J. 1993; 7: 1330-1337Crossref PubMed Scopus (195) Google Scholar). They may also be important for maintaining protein conformation, enzymatic activity, and other structural functions (15Opdenakker G. Rudd P.M. Ponting C.P. Dwek R.A. FASEB J. 1993; 7: 1330-1337Crossref PubMed Scopus (195) Google Scholar,16Imperiali B. Rickert K.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 97-101Crossref PubMed Scopus (158) Google Scholar). Among GPCRs, the role of carbohydrate moieties is somewhat less clear, with variable effects on ligand binding, signal transduction, and cell surface expression (17Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (191) Google Scholar, 18Ding D.X.-H. Vera J.C. Heaney M.L. Golde D.W. J. Biol. Chem. 1995; 270: 24580-24584Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 19Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar, 20Rodriguez C.G. Cundell D.R. Tuomanen E.I. Kolakowski Jr., L.F. Gerard C. Gerard N.P. J. Biol. Chem. 1995; 270: 25178-25184Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar, 22Liu X. Davis D. Segaloff D.L. J. Biol. Chem. 1993; 268: 1513-1516Abstract Full Text PDF PubMed Google Scholar, 23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). The function ofN-linked glycosylation for the CaR or other members of this unique GPCR subfamily with significantly larger ECDs and a greater number of putative N-linked glycosylation sites has not been studied extensively. Our previous study with tunicamycin indicated that inhibition of N-linked glycosylation blocks normal cell surface expression of the hCaR (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar). However, this study did not permit determination of the precise number and location of N-linked glycosylation sites, nor did it allow us to define distinctive roles, if any, of specific sites in the processing, cell surface expression, and signal transduction of the hCaR. To address these questions, we mutated the potential N-linked glycosylation sites of the hCaR, substituting glutamine for asparagine to disrupt glycosylation, and transfected the mutant receptor cDNAs into HEK-293 cells to analyze the effects on expression and function.DISCUSSIONThe importance of N-linked glycosylation for expression and function has been extensively studied for many GPCRs but not for the unique subset to which the CaR belongs. Most GPCRs of the large rhodopsin subfamily that includes adrenergic receptors and a number of peptide receptors have relatively short extracellular amino termini with one or two glycosylation sites. In some (17Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (191) Google Scholar, 18Ding D.X.-H. Vera J.C. Heaney M.L. Golde D.W. J. Biol. Chem. 1995; 270: 24580-24584Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Rodriguez C.G. Cundell D.R. Tuomanen E.I. Kolakowski Jr., L.F. Gerard C. Gerard N.P. J. Biol. Chem. 1995; 270: 25178-25184Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) but not all (19Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar) cases, glycosylation of at least one site is required for efficient cell surface expression. The glycoprotein hormone receptors have a large amino-terminal ECD, approximately two-thirds the size of the CaR, with three (follicle-stimulating hormone) or six (luteinizing hormone, thyroid-stimulating hormone) putative glycosylation sites (21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar,23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar, 30Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (75) Google Scholar). For both the follicle-stimulating hormone and thyroid-stimulating hormone receptors, glycosylation of a specific subset of sites (one of three for follicle-stimulating hormone and two of six for thyroid-stimulating hormone) was shown to be absolutely required for cell surface expression (21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar, 23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). For the follicle-stimulating hormone receptor, enzymatic deglycosylation studies of wild type receptor showed that carbohydrate is not, however, essential for hormone binding (23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). Results for the luteinizing hormone receptor are controversial. One study concluded that glycosylation at two of six sites is required for proper receptor folding but not for hormone binding per se (31Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Crossref PubMed Scopus (79) Google Scholar). A different study employing tunicamycin to inhibit glycosylation suggested that carbohydrate is not required at all for proper receptor folding and hormone binding (30Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (75) Google Scholar). Loss of function observed in other mutagenesis studies was attributed to differences in primary sequence rather than disruption of glycosylation.Previous studies on the CaR indicated that it is relatively heavily glycosylated (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar) and that inhibition of glycosylation with tunicamycin blocks receptor expression at the cell surface (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar). The present studies were directed at defining which of the 11 putative glycosylation sites in the ECD are actually used and the role of glycosylation in receptor expression and function. Based on studies of a series of mutants in which glycosylation consensus site asparagines were changed to glutamine singly and in various combinations, we identified eight of the 11 putative sites as containingN-linked sugar. Disruption of any of these eight sites alone caused a slight increase in mobility of the expressed receptor on SDS-PAGE; disruption of tandem sites caused additive changes in mobility. Glycosidase digestion of mutants in which all but one of these eight sites were disrupted confirmed that each of these sites could be glycosylated. The remaining three sites in contrast did not appear to be glycosylated unless the other eight sites were disrupted, suggesting that they are far less efficiently modified. This overall pattern, obtained in HEK-293 cells transiently transfected, may not exactly reflect the pattern of glycosylation of CaRs expressed endogenously in tissues such as parathyroid and kidney, but the similarity in size of bands obtained in immunoblots of membranes from transfected 293 cells and from parathyroid (26Goldsmith P.K. Fan G. Miller J.L. Rogers K.V. Spiegel A.M. J. Bone Miner. Res. 1997; 12: 1780-1788Crossref PubMed Scopus (45) Google Scholar, 28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) suggests that the glycosylation pattern is likely to be quite similar.The principal functional effect of disruption of CaR glycosylation sites we were able to define is loss of cell surface expression. Elimination of one or two of the employed glycosylation sites did not measurably impair expression. The disease familial hypocalciuric hypercalcemia is caused by loss of function mutations of the hCaR (1Brown E.M. Pollak M. Hebert S.C. Annu. Rev. Med. 1998; 49: 15-29Crossref PubMed Scopus (190) Google Scholar). Many single amino acid mutations have been identified that cause this disease (28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) but none to date that involve mutation of a consensus glycosylation site, consistent with the lack of significant functional impairment of disruption of single glycosylation sites. Elimination of three or more glycosylation sites caused progressive reduction in cell surface expression. Elimination of five or more sites led to a form of the receptor characterized by a single, Endo H-sensitive band on immunoblots. Previous studies of other CaR mutants have indicated that this band probably represents a form of the CaR that is misfolded, retained in the endoplasmic reticulum, and unable to undergo normal processing in the Golgi needed for cell surface expression (25Ray K. Fan G.-F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Immunofluorescence experiments and cell surface protein labeling with biotin confirmed that the Endo-H-sensitive band seen on immunoblots corresponds to an intracellular form of the hCaR.We interpret the present studies to indicate a critical role forN-linked glycosylation of a minimum number of CaR ECD sites in normal folding of the receptor as it is being synthesized in the endoplasmic reticulum. Interestingly, the degree of functional impairment differed with the site of disruption of glycosylation. The N1–5Q mutant showed more severe loss of expression than did the N4–8Q mutant; although five sites are disrupted in both mutants, apparently the loss of more amino-terminal sites has a greater impact on protein folding and retention of the receptor in the endoplasmic reticulum. All of these results must be qualified by the caveat that effects of asparagine to glutamine substitution, although a conservative change, could be due to altered primary sequence rather than disruption of glycosylation. The fact that mutation at single and even double sites did not impair function makes this concern less likely. Also, the ability of tunicamycin to inhibit cell surface expression of the receptor supports a key role for glycosylation in receptor folding and trafficking (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar).Any attempt to study the impact of disruption of glycosylation of the CaR on signal transduction is limited by the lack of a ligand binding assay and the requirement of cell surface expression for measurement of extracellular calcium activation of the receptor in the PI hydrolysis assay. Thus, our studies cannot exclude a role for carbohydrate in calcium binding to and activation of the receptor. Nonetheless, the ability of various glycosylation-deficient mutants to respond to calcium appeared to be impaired in proportion to their reduction in cell surface expression. Mutants such as N1–4Q and N5–8Q when expressed at levels comparable with wild type showed roughly equivalent extracellular calcium-stimulated PI hydrolysis to the wild type CaR, suggesting that a substantial lack of carbohydrate in the ECD does not impair receptor signaling. More definitive studies including enzymatic deglycosylation of purified CaR combined with direct measures of calcium activation will be required to elucidate the role, if any, of ECD carbohydrate in receptor function.The present studies have defined an important role for specific glycosylation sites in CaR processing and cell surface expression. These studies have identified mutant forms of the receptor with multiple glycosylation sites disrupted that nonetheless are able to be expressed as functional receptors at the cell surface. Such mutants may facilitate expression and purification of a form of the CaR ECD useful for x-ray crystallographic studies needed to define the three-dimensional structure of this unique receptor domain. The calcium receptor (CaR)1 is a G-protein-coupled-receptor (GPCR) involved in extracellular calcium homeostasis by controlling the rate of parathyroid hormone secretion from the parathyroid gland and the rate of calcium reabsorbtion by the kidney (1Brown E.M. Pollak M. Hebert S.C. Annu. Rev. Med. 1998; 49: 15-29Crossref PubMed Scopus (190) Google Scholar). Recent evidence suggests that the CaR is also involved in diverse cellular responses to extracellular calcium within microenvironments in other organs such as brain, skin, bone, and intestine (2Chattopadhyay N. Vassilev P.M. Brown E.M. Biol. Chem. 1997; 378: 759-768PubMed Google Scholar). Extracellular calcium ion ([Ca2+]o) activates the CaR, leading to activation of phospholipase-Cβ via the Gqsubfamily of G-proteins; this increases phosphoinositide (PI) hydrolysis, which in turn causes release of calcium from intracellular stores (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar). The CaR, metabotropic glutamate receptors (4Nakanishi S. Science. 1992; 258: 597-603Crossref PubMed Scopus (2284) Google Scholar), and a subgroup of putative pheromone receptors in the vomeronasal organ (5Herrada G. Dulac C. Cell. 1997; 90: 763-773Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 6Matsunami H. Buck L.B. Cell. 1997; 90: 775-784Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar, 7Ryba N.J. Tirindelli R. Neuron. 1997; 19: 371-379Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar) comprise a unique subset of the superfamily of GPCRs characterized by a relatively large (500–600-residue) amino-terminal extracellular domain (ECD). The ECD of the human CaR (hCaR) (8Garrett J.E. Capuano I.V. Hammerland L.G. Hung B.C.P. Brown E.M. Hebert S.C. Nemeth E.F. Fuller F. J Biol. Chem. 1995; 270: 12919-12925Crossref PubMed Scopus (455) Google Scholar) contains 11 potentialN-linked glycosylation sites (9Shakin-Eshleman S.H. Spitalnik S.L. Kasturi L. J. Biol. Chem. 1996; 271: 6363-6366Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), of which nine are highly conserved in bovine (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar), rat (10Riccardi D. Park J. Lee W.S. Gamba G. Brown E.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (434) Google Scholar, 11Ruat M. Molliver M.E. Snowman A.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3161-3165Crossref PubMed Scopus (343) Google Scholar), rabbit (12Butters Jr., R.R. Chattopadhyay N. Nielsen P. Smith C.P. Mithal A. Kifor O. Bai M. Quinn S. Goldsmith P. Hurwitz S. Krapcho K. Busby J. Brown E.M. J Bone Miner. Res. 1997; 12: 568-579Crossref PubMed Scopus (63) Google Scholar), and chicken (13Diaz R. Hurwitz S. Chattopadhyay N. Pines M. Yang Y. Kifor O. Einat M.S. Butters R. Hebert S.C. Brown E.M. Am. J. Physiol. 1997; 273: R1008-R1016Crossref PubMed Google Scholar) CaRs. The remaining two sites are conserved among human, rabbit, and chicken CaRs, but only one of the two (Asn-386 or -400 in the hCaR sequence) is conserved in rat and bovine CaRs, respectively. The carbohydrate moieties of glycoproteins in general are believed to be important for facilitating protein folding, protection from proteolysis, intracellular trafficking, secretion, and cell surface expression (14Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (558) Google Scholar, 15Opdenakker G. Rudd P.M. Ponting C.P. Dwek R.A. FASEB J. 1993; 7: 1330-1337Crossref PubMed Scopus (195) Google Scholar). They may also be important for maintaining protein conformation, enzymatic activity, and other structural functions (15Opdenakker G. Rudd P.M. Ponting C.P. Dwek R.A. FASEB J. 1993; 7: 1330-1337Crossref PubMed Scopus (195) Google Scholar,16Imperiali B. Rickert K.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 97-101Crossref PubMed Scopus (158) Google Scholar). Among GPCRs, the role of carbohydrate moieties is somewhat less clear, with variable effects on ligand binding, signal transduction, and cell surface expression (17Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (191) Google Scholar, 18Ding D.X.-H. Vera J.C. Heaney M.L. Golde D.W. J. Biol. Chem. 1995; 270: 24580-24584Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 19Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar, 20Rodriguez C.G. Cundell D.R. Tuomanen E.I. Kolakowski Jr., L.F. Gerard C. Gerard N.P. J. Biol. Chem. 1995; 270: 25178-25184Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar, 22Liu X. Davis D. Segaloff D.L. J. Biol. Chem. 1993; 268: 1513-1516Abstract Full Text PDF PubMed Google Scholar, 23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). The function ofN-linked glycosylation for the CaR or other members of this unique GPCR subfamily with significantly larger ECDs and a greater number of putative N-linked glycosylation sites has not been studied extensively. Our previous study with tunicamycin indicated that inhibition of N-linked glycosylation blocks normal cell surface expression of the hCaR (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar). However, this study did not permit determination of the precise number and location of N-linked glycosylation sites, nor did it allow us to define distinctive roles, if any, of specific sites in the processing, cell surface expression, and signal transduction of the hCaR. To address these questions, we mutated the potential N-linked glycosylation sites of the hCaR, substituting glutamine for asparagine to disrupt glycosylation, and transfected the mutant receptor cDNAs into HEK-293 cells to analyze the effects on expression and function. DISCUSSIONThe importance of N-linked glycosylation for expression and function has been extensively studied for many GPCRs but not for the unique subset to which the CaR belongs. Most GPCRs of the large rhodopsin subfamily that includes adrenergic receptors and a number of peptide receptors have relatively short extracellular amino termini with one or two glycosylation sites. In some (17Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (191) Google Scholar, 18Ding D.X.-H. Vera J.C. Heaney M.L. Golde D.W. J. Biol. Chem. 1995; 270: 24580-24584Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Rodriguez C.G. Cundell D.R. Tuomanen E.I. Kolakowski Jr., L.F. Gerard C. Gerard N.P. J. Biol. Chem. 1995; 270: 25178-25184Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) but not all (19Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar) cases, glycosylation of at least one site is required for efficient cell surface expression. The glycoprotein hormone receptors have a large amino-terminal ECD, approximately two-thirds the size of the CaR, with three (follicle-stimulating hormone) or six (luteinizing hormone, thyroid-stimulating hormone) putative glycosylation sites (21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar,23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar, 30Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (75) Google Scholar). For both the follicle-stimulating hormone and thyroid-stimulating hormone receptors, glycosylation of a specific subset of sites (one of three for follicle-stimulating hormone and two of six for thyroid-stimulating hormone) was shown to be absolutely required for cell surface expression (21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar, 23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). For the follicle-stimulating hormone receptor, enzymatic deglycosylation studies of wild type receptor showed that carbohydrate is not, however, essential for hormone binding (23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). Results for the luteinizing hormone receptor are controversial. One study concluded that glycosylation at two of six sites is required for proper receptor folding but not for hormone binding per se (31Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Crossref PubMed Scopus (79) Google Scholar). A different study employing tunicamycin to inhibit glycosylation suggested that carbohydrate is not required at all for proper receptor folding and hormone binding (30Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (75) Google Scholar). Loss of function observed in other mutagenesis studies was attributed to differences in primary sequence rather than disruption of glycosylation.Previous studies on the CaR indicated that it is relatively heavily glycosylated (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar) and that inhibition of glycosylation with tunicamycin blocks receptor expression at the cell surface (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar). The present studies were directed at defining which of the 11 putative glycosylation sites in the ECD are actually used and the role of glycosylation in receptor expression and function. Based on studies of a series of mutants in which glycosylation consensus site asparagines were changed to glutamine singly and in various combinations, we identified eight of the 11 putative sites as containingN-linked sugar. Disruption of any of these eight sites alone caused a slight increase in mobility of the expressed receptor on SDS-PAGE; disruption of tandem sites caused additive changes in mobility. Glycosidase digestion of mutants in which all but one of these eight sites were disrupted confirmed that each of these sites could be glycosylated. The remaining three sites in contrast did not appear to be glycosylated unless the other eight sites were disrupted, suggesting that they are far less efficiently modified. This overall pattern, obtained in HEK-293 cells transiently transfected, may not exactly reflect the pattern of glycosylation of CaRs expressed endogenously in tissues such as parathyroid and kidney, but the similarity in size of bands obtained in immunoblots of membranes from transfected 293 cells and from parathyroid (26Goldsmith P.K. Fan G. Miller J.L. Rogers K.V. Spiegel A.M. J. Bone Miner. Res. 1997; 12: 1780-1788Crossref PubMed Scopus (45) Google Scholar, 28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) suggests that the glycosylation pattern is likely to be quite similar.The principal functional effect of disruption of CaR glycosylation sites we were able to define is loss of cell surface expression. Elimination of one or two of the employed glycosylation sites did not measurably impair expression. The disease familial hypocalciuric hypercalcemia is caused by loss of function mutations of the hCaR (1Brown E.M. Pollak M. Hebert S.C. Annu. Rev. Med. 1998; 49: 15-29Crossref PubMed Scopus (190) Google Scholar). Many single amino acid mutations have been identified that cause this disease (28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) but none to date that involve mutation of a consensus glycosylation site, consistent with the lack of significant functional impairment of disruption of single glycosylation sites. Elimination of three or more glycosylation sites caused progressive reduction in cell surface expression. Elimination of five or more sites led to a form of the receptor characterized by a single, Endo H-sensitive band on immunoblots. Previous studies of other CaR mutants have indicated that this band probably represents a form of the CaR that is misfolded, retained in the endoplasmic reticulum, and unable to undergo normal processing in the Golgi needed for cell surface expression (25Ray K. Fan G.-F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Immunofluorescence experiments and cell surface protein labeling with biotin confirmed that the Endo-H-sensitive band seen on immunoblots corresponds to an intracellular form of the hCaR.We interpret the present studies to indicate a critical role forN-linked glycosylation of a minimum number of CaR ECD sites in normal folding of the receptor as it is being synthesized in the endoplasmic reticulum. Interestingly, the degree of functional impairment differed with the site of disruption of glycosylation. The N1–5Q mutant showed more severe loss of expression than did the N4–8Q mutant; although five sites are disrupted in both mutants, apparently the loss of more amino-terminal sites has a greater impact on protein folding and retention of the receptor in the endoplasmic reticulum. All of these results must be qualified by the caveat that effects of asparagine to glutamine substitution, although a conservative change, could be due to altered primary sequence rather than disruption of glycosylation. The fact that mutation at single and even double sites did not impair function makes this concern less likely. Also, the ability of tunicamycin to inhibit cell surface expression of the receptor supports a key role for glycosylation in receptor folding and trafficking (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar).Any attempt to study the impact of disruption of glycosylation of the CaR on signal transduction is limited by the lack of a ligand binding assay and the requirement of cell surface expression for measurement of extracellular calcium activation of the receptor in the PI hydrolysis assay. Thus, our studies cannot exclude a role for carbohydrate in calcium binding to and activation of the receptor. Nonetheless, the ability of various glycosylation-deficient mutants to respond to calcium appeared to be impaired in proportion to their reduction in cell surface expression. Mutants such as N1–4Q and N5–8Q when expressed at levels comparable with wild type showed roughly equivalent extracellular calcium-stimulated PI hydrolysis to the wild type CaR, suggesting that a substantial lack of carbohydrate in the ECD does not impair receptor signaling. More definitive studies including enzymatic deglycosylation of purified CaR combined with direct measures of calcium activation will be required to elucidate the role, if any, of ECD carbohydrate in receptor function.The present studies have defined an important role for specific glycosylation sites in CaR processing and cell surface expression. These studies have identified mutant forms of the receptor with multiple glycosylation sites disrupted that nonetheless are able to be expressed as functional receptors at the cell surface. Such mutants may facilitate expression and purification of a form of the CaR ECD useful for x-ray crystallographic studies needed to define the three-dimensional structure of this unique receptor domain. The importance of N-linked glycosylation for expression and function has been extensively studied for many GPCRs but not for the unique subset to which the CaR belongs. Most GPCRs of the large rhodopsin subfamily that includes adrenergic receptors and a number of peptide receptors have relatively short extracellular amino termini with one or two glycosylation sites. In some (17Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (191) Google Scholar, 18Ding D.X.-H. Vera J.C. Heaney M.L. Golde D.W. J. Biol. Chem. 1995; 270: 24580-24584Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Rodriguez C.G. Cundell D.R. Tuomanen E.I. Kolakowski Jr., L.F. Gerard C. Gerard N.P. J. Biol. Chem. 1995; 270: 25178-25184Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) but not all (19Innamorati G. Sadeghi H. Birnbaumer M. Mol. Pharmacol. 1996; 50: 467-473PubMed Google Scholar) cases, glycosylation of at least one site is required for efficient cell surface expression. The glycoprotein hormone receptors have a large amino-terminal ECD, approximately two-thirds the size of the CaR, with three (follicle-stimulating hormone) or six (luteinizing hormone, thyroid-stimulating hormone) putative glycosylation sites (21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar,23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar, 30Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (75) Google Scholar). For both the follicle-stimulating hormone and thyroid-stimulating hormone receptors, glycosylation of a specific subset of sites (one of three for follicle-stimulating hormone and two of six for thyroid-stimulating hormone) was shown to be absolutely required for cell surface expression (21Russo D. Chazenbalk G.D. Nagayama Y. Wadsworth H.L. Rapoport B. Mol. Endocrinol. 1991; 5: 29-33Crossref PubMed Scopus (89) Google Scholar, 23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). For the follicle-stimulating hormone receptor, enzymatic deglycosylation studies of wild type receptor showed that carbohydrate is not, however, essential for hormone binding (23Davis D. Liu X. Segaloff D.L. Mol. Endocrinol. 1995; 9: 159-170Crossref PubMed Google Scholar). Results for the luteinizing hormone receptor are controversial. One study concluded that glycosylation at two of six sites is required for proper receptor folding but not for hormone binding per se (31Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Crossref PubMed Scopus (79) Google Scholar). A different study employing tunicamycin to inhibit glycosylation suggested that carbohydrate is not required at all for proper receptor folding and hormone binding (30Davis D.P. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1997; 11: 550-562Crossref PubMed Scopus (75) Google Scholar). Loss of function observed in other mutagenesis studies was attributed to differences in primary sequence rather than disruption of glycosylation. Previous studies on the CaR indicated that it is relatively heavily glycosylated (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2344) Google Scholar) and that inhibition of glycosylation with tunicamycin blocks receptor expression at the cell surface (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar). The present studies were directed at defining which of the 11 putative glycosylation sites in the ECD are actually used and the role of glycosylation in receptor expression and function. Based on studies of a series of mutants in which glycosylation consensus site asparagines were changed to glutamine singly and in various combinations, we identified eight of the 11 putative sites as containingN-linked sugar. Disruption of any of these eight sites alone caused a slight increase in mobility of the expressed receptor on SDS-PAGE; disruption of tandem sites caused additive changes in mobility. Glycosidase digestion of mutants in which all but one of these eight sites were disrupted confirmed that each of these sites could be glycosylated. The remaining three sites in contrast did not appear to be glycosylated unless the other eight sites were disrupted, suggesting that they are far less efficiently modified. This overall pattern, obtained in HEK-293 cells transiently transfected, may not exactly reflect the pattern of glycosylation of CaRs expressed endogenously in tissues such as parathyroid and kidney, but the similarity in size of bands obtained in immunoblots of membranes from transfected 293 cells and from parathyroid (26Goldsmith P.K. Fan G. Miller J.L. Rogers K.V. Spiegel A.M. J. Bone Miner. Res. 1997; 12: 1780-1788Crossref PubMed Scopus (45) Google Scholar, 28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) suggests that the glycosylation pattern is likely to be quite similar. The principal functional effect of disruption of CaR glycosylation sites we were able to define is loss of cell surface expression. Elimination of one or two of the employed glycosylation sites did not measurably impair expression. The disease familial hypocalciuric hypercalcemia is caused by loss of function mutations of the hCaR (1Brown E.M. Pollak M. Hebert S.C. Annu. Rev. Med. 1998; 49: 15-29Crossref PubMed Scopus (190) Google Scholar). Many single amino acid mutations have been identified that cause this disease (28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) but none to date that involve mutation of a consensus glycosylation site, consistent with the lack of significant functional impairment of disruption of single glycosylation sites. Elimination of three or more glycosylation sites caused progressive reduction in cell surface expression. Elimination of five or more sites led to a form of the receptor characterized by a single, Endo H-sensitive band on immunoblots. Previous studies of other CaR mutants have indicated that this band probably represents a form of the CaR that is misfolded, retained in the endoplasmic reticulum, and unable to undergo normal processing in the Golgi needed for cell surface expression (25Ray K. Fan G.-F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 28Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Immunofluorescence experiments and cell surface protein labeling with biotin confirmed that the Endo-H-sensitive band seen on immunoblots corresponds to an intracellular form of the hCaR. We interpret the present studies to indicate a critical role forN-linked glycosylation of a minimum number of CaR ECD sites in normal folding of the receptor as it is being synthesized in the endoplasmic reticulum. Interestingly, the degree of functional impairment differed with the site of disruption of glycosylation. The N1–5Q mutant showed more severe loss of expression than did the N4–8Q mutant; although five sites are disrupted in both mutants, apparently the loss of more amino-terminal sites has a greater impact on protein folding and retention of the receptor in the endoplasmic reticulum. All of these results must be qualified by the caveat that effects of asparagine to glutamine substitution, although a conservative change, could be due to altered primary sequence rather than disruption of glycosylation. The fact that mutation at single and even double sites did not impair function makes this concern less likely. Also, the ability of tunicamycin to inhibit cell surface expression of the receptor supports a key role for glycosylation in receptor folding and trafficking (24Fan G. Goldsmith P.K. Collins R. Dunn C.K. Krapcho K.J. Rogers K.V. Spiegel A.M. Endocrinology. 1997; 138: 1916-1922Crossref PubMed Google Scholar). Any attempt to study the impact of disruption of glycosylation of the CaR on signal transduction is limited by the lack of a ligand binding assay and the requirement of cell surface expression for measurement of extracellular calcium activation of the receptor in the PI hydrolysis assay. Thus, our studies cannot exclude a role for carbohydrate in calcium binding to and activation of the receptor. Nonetheless, the ability of various glycosylation-deficient mutants to respond to calcium appeared to be impaired in proportion to their reduction in cell surface expression. Mutants such as N1–4Q and N5–8Q when expressed at levels comparable with wild type showed roughly equivalent extracellular calcium-stimulated PI hydrolysis to the wild type CaR, suggesting that a substantial lack of carbohydrate in the ECD does not impair receptor signaling. More definitive studies including enzymatic deglycosylation of purified CaR combined with direct measures of calcium activation will be required to elucidate the role, if any, of ECD carbohydrate in receptor function. The present studies have defined an important role for specific glycosylation sites in CaR processing and cell surface expression. These studies have identified mutant forms of the receptor with multiple glycosylation sites disrupted that nonetheless are able to be expressed as functional receptors at the cell surface. Such mutants may facilitate expression and purification of a form of the CaR ECD useful for x-ray crystallographic studies needed to define the three-dimensional structure of this unique receptor domain. We thank Dr. Regina Collins for assistance with cell culture procedures and Mandy Wang, a summer student from the University of Arizona, Tucson, for excellent technical assistance.
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