Structurally Diverse N-terminal Peptides of Parathyroid Hormone (PTH) and PTH-related Peptide (PTHRP) Inhibit the Na+/H+ Exchanger NHE3 Isoform by Binding to the PTH/PTHRP Receptor Type I and Activating Distinct Signaling Pathways
1996; Elsevier BV; Volume: 271; Issue: 25 Linguagem: Inglês
10.1074/jbc.271.25.14931
ISSN1083-351X
AutoresArezou Azarani, David Goltzman, John Orlowski,
Tópico(s)Thyroid Disorders and Treatments
ResumoN-terminal peptides of parathyroid hormone (PTH) and PTH-related peptide (PTHRP) elicit a wide variety of biological responses in target cells, including the inhibition of Na+/H+ exchanger NHE3 activity in renal cells. This response is believed to be mediated by ligand binding to a common receptor (i.e. PTH/PTHRP receptor type I) and activation of cAMP-dependent and/or Ca2+/phospholipid-dependent protein kinases (PKA and PKC, respectively). However, the mechanism of action of these N-terminal peptides is now unclear because of recent data reporting the existence of additional receptor isoforms. Therefore, to directly examine the ligand binding and signaling characteristics of the PTH/PTHRP receptor type I and its ability to elicit a biological response, cDNAs encoding the rat type I receptor and the rat NHE3 isoform were transfected into Chinese hamster ovary (AP-1) cells that lack endogenous expression of these proteins. Competition binding assays using [125I-Tyr36]PTHRP-(1–36)-NH2 radioligand indicated that several biologically active human N-terminal PTH and PTHRP fragments (PTH-(1–34), PTH-(3–34), PTH-(28–42), PTH-(28–48), and PTHRP-(1–34)) were capable of binding to the type I receptor. Both PTH-(1–34) and PTHRP-(1–34) stimulated adenylate cyclase and PKC activities in these cells, whereas PTH-(3–34), PTH-(28–42), and PTH-(28–48) selectively enhanced only PKC activity. PTHRP-(1–16), a biologically inert fragment, was incapable of binding to this receptor and influencing either the PKA or PKC pathway. Furthermore, all the analogues with the exception of PTHRP-(1–16) inhibited NHE3 activity. Inhibition of PKC by the potent antagonist chelerythrine chloride abolished the depression of NHE3 activity by PTH-(3–34), PTH-(28–42), and PTH-(28–48) but did not alleviate the effects of PTH-(1–34). Likewise, antagonism of PKA by H-89 was unable to prevent the inhibition caused by PTH-(1–34). However, inhibition of both PKA and PKC by the nonselective protein kinase antagonist H-7 abolished the reduction of NHE3 activity by PTH-(1–34). These data indicate that discrete N-terminal analogues of PTH and PTHRP can interact with the classical PTH/PTHRP receptor type I and activate PKA and/or PKC. Activation of either signaling pathway independently leads to inhibition of NHE3. N-terminal peptides of parathyroid hormone (PTH) and PTH-related peptide (PTHRP) elicit a wide variety of biological responses in target cells, including the inhibition of Na+/H+ exchanger NHE3 activity in renal cells. This response is believed to be mediated by ligand binding to a common receptor (i.e. PTH/PTHRP receptor type I) and activation of cAMP-dependent and/or Ca2+/phospholipid-dependent protein kinases (PKA and PKC, respectively). However, the mechanism of action of these N-terminal peptides is now unclear because of recent data reporting the existence of additional receptor isoforms. Therefore, to directly examine the ligand binding and signaling characteristics of the PTH/PTHRP receptor type I and its ability to elicit a biological response, cDNAs encoding the rat type I receptor and the rat NHE3 isoform were transfected into Chinese hamster ovary (AP-1) cells that lack endogenous expression of these proteins. Competition binding assays using [125I-Tyr36]PTHRP-(1–36)-NH2 radioligand indicated that several biologically active human N-terminal PTH and PTHRP fragments (PTH-(1–34), PTH-(3–34), PTH-(28–42), PTH-(28–48), and PTHRP-(1–34)) were capable of binding to the type I receptor. Both PTH-(1–34) and PTHRP-(1–34) stimulated adenylate cyclase and PKC activities in these cells, whereas PTH-(3–34), PTH-(28–42), and PTH-(28–48) selectively enhanced only PKC activity. PTHRP-(1–16), a biologically inert fragment, was incapable of binding to this receptor and influencing either the PKA or PKC pathway. Furthermore, all the analogues with the exception of PTHRP-(1–16) inhibited NHE3 activity. Inhibition of PKC by the potent antagonist chelerythrine chloride abolished the depression of NHE3 activity by PTH-(3–34), PTH-(28–42), and PTH-(28–48) but did not alleviate the effects of PTH-(1–34). Likewise, antagonism of PKA by H-89 was unable to prevent the inhibition caused by PTH-(1–34). However, inhibition of both PKA and PKC by the nonselective protein kinase antagonist H-7 abolished the reduction of NHE3 activity by PTH-(1–34). These data indicate that discrete N-terminal analogues of PTH and PTHRP can interact with the classical PTH/PTHRP receptor type I and activate PKA and/or PKC. Activation of either signaling pathway independently leads to inhibition of NHE3. INTRODUCTIONParathyroid hormone (PTH), 1The abbreviations used are: PTHparathyroid hormonePTHRPPTH-related peptidePKAprotein kinase APKCprotein kinase CDTTdithiothreitolPMSFphenylmethylsulfonyl fluoride; α-MEM. α-minimal essential mediumPMAphorbol 12-myristate 13-acetateBCECF2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein. PTH-related peptide (PTHRP), and their N-terminal analogues influence plasma calcium and phosphate homeostasis by regulating a variety of membrane ion channels and transporters within target cells, including Ca2+ channels (1Yamaguihi D.T. Hahn T.J. Iida-Klein A. Kleeman C.R. Muallem S. J. Biol. Chem. 1987; 262: 7711-7718Google Scholar), Cl− channels (2Chesnoy-Marchais D. Fritsch J. Pflügers Arch. 1989; 415: 104-114Google Scholar), Na+/Pi cotransporters (3Malmström K. Stange G. Murer H. Biochem. J. 1988; 251: 207-213Google Scholar, 4Bringhurst F.R. Jüppner H. Guo J. Ureña P. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.-B. Segre G.V. Endocrinology. 1993; 132: 2090-2098Google Scholar), and Na+/H+ exchangers (5Pollock A.S. Warnock D.G. Strewler G.J. Am. J. Physiol. 1986; 250: F217-F225Google Scholar, 6Helmle-Kolb C. Montrose M.H. Stange G. Murer H. Pflügers Arch. 1990; 415: 461-470Google Scholar, 7Azarani A. Goltzman D. Orlowski J. J. Biol. Chem. 1995; 270: 20004-20010Google Scholar, 8Azarani A. Orlowski J. Goltzman D. J. Biol. Chem. 1995; 270: 23166-23172Google Scholar). This multiplicity of actions is generally believed to reflect ligand binding to a common heterotrimeric G-protein-coupled receptor that is linked to multiple effector systems (i.e. adenylate cyclase and phospholipase C) (9Abou-Samra A.-B. Jüppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Ureña P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Google Scholar). This is exemplified in renal proximal tubule OK cells where N-terminal peptide fragments of PTH and PTHRP (i.e. PTH-(1–34), PTH-(3–34), PTH-(28–42), PTH-(28–48), and PTHRP-(1–34)) rapidly inhibit the activity of the apically localized Na+/H+ exchanger NHE3 isoform by a mechanism involving cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) (7Azarani A. Goltzman D. Orlowski J. J. Biol. Chem. 1995; 270: 20004-20010Google Scholar). This regulation is remarkable given that PTH (an 84-amino acid peptide) and PTHRP (a 139–173-amino acid peptide depending on the species) (10Orloff J.J. Wu T.L. Stewart A.F. Endocr. Rev. 1989; 10: 476-495Google Scholar) share minimal identity in primary structure, with only 8 of the 13 N-terminal amino acids being common between these two peptides. PTH and PTHRP require the first two N-terminal amino acids and amino acids 25–34 to stimulate adenylate cyclase activity (11Goltzman D. Peytremann A. Callahan E. Tregear G.W. Potts Jr., J.T. J. Biol. Chem. 1975; 250: 3199-3203Google Scholar, 12Rabbani S.A. Kaiser S.M. Henderson J.E. Bernier S.M. Mouland A.J. Roy D.R. Zahab D.M. Sung W.L. Goltzman D. Hendy G.N. Biochemistry. 1990; 29: 10080-10089Google Scholar, 13Rabbani S.A. Mitchell J. Roy D.R. Hendy G.N. Goltzman D. Endocrinology. 1988; 123: 2709-2716Google Scholar, 14Rosenblatt M. N. Engl. J. Med. 1986; 315: 1004-1013Google Scholar). In contrast, amino acids 3–34 and even smaller regions (amino acids 28–34) appear sufficient to activate PKC translocation to the plasma membrane (15Fujimori A. Cheng S.L. Avioli L.V. Civitelli R. Endocrinology. 1992; 130: 29-36Google Scholar, 16Jouishomme H. Whitfield J.F. Chakravarthy B. Durkin J.P. Gagnon L. Isaacs R.J. Maclean S. Neugebauer W. Willick G. Rixon R.H. Endocrinology. 1992; 130: 53-60Google Scholar, 17Jouishomme H. Whitfield J.F. Gagnon L. Maclean S. Isaacs R. Chakravarthy B. Durkin J. Neugebauer W. Willick G. Rixon R.H. J. Bone Miner. Res. 1994; 9: 943-949Google Scholar).However, the universality of this signaling paradigm to account for the diverse actions of N-terminal analogues of PTH and PTHRP is no longer tenable in view of recent data reporting the existence of additional related receptors. Usdin et al. (18Usdin T.B. Gruber C. Bonner T.I. J. Biol. Chem. 1995; 270: 15455-15458Google Scholar) recently isolated and characterized a unique receptor (called PTH2) from a rat brain cDNA library that shares 70% amino acid identity to the classical PTH/PTHRP receptor (type I). PTH2 mRNA is expressed predominantly in brain and pancreas, and to a much lesser extent in placenta and testis. PTH2 is also functionally distinguished from the type I receptor by its selective binding of PTH and by its potent activation of adenylate cyclase activity.In addition, biochemical studies in normal keratinocytes and squamous carcinoma cell lines suggest the existence of a novel PTH/PTHRP receptor (type II) that differs qualitatively in its intracellular signaling properties from those of the type I receptor and PTH2 (19Orloff J.J. Kats Y. Ureña P. Schipani E. Vasavada R.C. Philbrick W.M. Behal A. Abou-Samra A.-B. Segre G.V. Jüppner H. Endocrinology. 1995; 136: 3016-3023Google Scholar). This potential receptor is activated by N-terminal peptide fragments of both PTH and PTHRP, leading to increases in intracellular Ca2+ but not cAMP. These cells also express multiple mRNA transcripts that hybridize to type I receptor cDNA probes, yet differ significantly in size from the type I receptor mRNA present in human bone SaOS-2 cells. Differently sized transcripts are also observed in rat kidney, liver, skin, and testes (20Ureña P. Kong X.F. Abou-Samra A.-B. Jüppner H. Kronenberg H.M. Potts Jr., J.T. Segre G.V. Endocrinology. 1993; 133: 617-623Google Scholar). These data have been interpreted to indicate the presence of a distinct gene product or an alternatively spliced variant of the type I receptor.Other circumstantial evidence also supports the existence of multiple receptors. A C-terminal peptide of human PTH (i.e. PTH-(53–84)) elicits a number of biological responses in rat osteosarcoma cells (21Murray T.M. Rao L.G. Muzaffar S.A. Ly H. Endocrinology. 1989; 124: 1097-1099Google Scholar, 22Kaji H. Sugimoto T. Kanatani M. Miyauchi A. Kimura T. Sakakibara S. Fukase M. Chihara K. Endocrinology. 1994; 134: 1897-1904Google Scholar) yet fails to bind to the human PTH/PTHRP receptor type I stably expressed in human embryonic kidney (HEK-293) cells (23Pines M. Adams A.E. Stueckle S. Bessalle R. Rashti-Behar V. Chorev M. Rosenblatt M. Suva L.J. Endocrinology. 1994; 135: 1713-1715Google Scholar). Thus, it is possible that the biological activity of some of these N- and C-terminal analogues is actually elicited by selective binding to other, as yet uncharacterized, PTH/PTHRP receptors. Understanding the signaling mechanism of these PTH/PTHRP analogues is of physiological relevance as parathyroid cells normally secrete peptide fragments of PTH (24MacGregor R.R. Jilka R.L. Hamilton J.W. J. Biol. Chem. 1986; 261: 1929-1934Google Scholar).In view of these data, we tested the hypothesis that N-terminal analogues of PTH and PTHRP can bind to and activate the PTH/PTHRP receptor type I, stimulate PKA and PKC, and acutely regulate the activity of the Na+/H+ exchanger NHE3 isoform, as is believed to occur in renal cells. This was accomplished by transient and stable transfection of cDNAs encoding the rat PTH/PTHRP receptor type I and the rat Na+/H+ exchanger NHE3 isoform into Chinese hamster ovary AP-1 cells that are devoid of endogenous Na+/H+ exchanger activity and lack responsiveness to PTH or PTHRP. The data clearly demonstrate that the AP-1-transfected cells were able to bind the various PTH and PTHRP analogues, stimulate production of multiple second messengers, and elicit biological responses in a manner that precisely mimics the responses observed in renal OK cells. Although the data do not exclude the presence of other PTH/PTHRP receptors in OK cells, it suggests that the PTH/PTHRP receptor type I is sufficient to mediate the diverse biological actions of these N-terminal PTH and PTHRP analogues in these cells.DISCUSSIONThe PTH/PTHRP receptor type I has been cloned from many tissues such as bone and kidney (9Abou-Samra A.-B. Jüppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Ureña P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Google Scholar, 37Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Google Scholar, 38Schipani E. Karga H. Karaplis A.C. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Abou-Samra A.-B. Jüppner H. Endocrinology. 1993; 132: 2157-2165Google Scholar, 39Pausova Z. Bourdon J. Clayton D. Mattei M.-G. Seldin M.F. Janicic N. Rivière M. Szpirer J. Levan G. Szpirer C. Goltzman D. Hendy G.N. Genomics. 1994; 20: 20-26Google Scholar) and belongs to a superfamily of G-protein-coupled receptors, including receptors for calcitonin, secretin, glucagon, glucagon-like peptide 1, growth hormone-releasing hormone, vasoactive intestinal peptide, gastric inhibitory peptide, corticotrophin-releasing factor A, and pituitary adenylate cyclase-activating peptide (40Segre G.V. Goldring S.R. Trends Endocrinol. Metab. 1993; 4: 309-314Google Scholar, 41Usdin T.B. Mezey E. Button D.C. Brownstein M.J. Bonner T.I. Endocrinology. 1993; 133: 2861-2870Google Scholar, 42Chen R. Lewis K. Perrin M. Vale W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8967-8971Google Scholar, 43Spengler D. Waeber C. Pantaloni C. Holsboer F. Bockaert J. Seeburg P.H. Journot L. Nature. 1993; 365: 170-175Google Scholar). Common features of these receptors include similar membrane topology (i.e. seven membrane-spanning segments) and the ability to activate G-proteins that modulate the adenylate cyclase-cAMP-PKA and/or phospholipase C-diacylglycerol-PKC pathways.Until recently, the ability of a single PTH/PTHRP receptor (i.e. type I) to couple to multiple effector systems was generally believed to account for the pleiotropic effects of PTH and PTHRP and their respective analogues in various target tissues (9Abou-Samra A.-B. Jüppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Ureña P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Google Scholar). However, this paradigm is no longer tenable following the discovery of a second receptor, PTH2, that is expressed primarily in brain and pancreas and is distinguished by its ability to bind only PTH and to activate the PKA pathway (18Usdin T.B. Gruber C. Bonner T.I. J. Biol. Chem. 1995; 270: 15455-15458Google Scholar).Other lines of investigation also indicate that additional PTH/PTHRP receptors may exist. 1) N-terminal analogues of PTH and PTHRP activate PKC activity in ROS 17/2 osteosarcoma cells in a biphasic manner, with one peak of activity obtained at low picomolar concentrations and the other at nanomolar concentrations (16Jouishomme H. Whitfield J.F. Chakravarthy B. Durkin J.P. Gagnon L. Isaacs R.J. Maclean S. Neugebauer W. Willick G. Rixon R.H. Endocrinology. 1992; 130: 53-60Google Scholar, 17Jouishomme H. Whitfield J.F. Gagnon L. Maclean S. Isaacs R. Chakravarthy B. Durkin J. Neugebauer W. Willick G. Rixon R.H. J. Bone Miner. Res. 1994; 9: 943-949Google Scholar, 44Gagnon L. Jouishomme H. Whitfield J.F. Durkin J.P. Maclean S. Neugebauer W. Willick G. Rixon R.H. Chakravarthy B. J. Bone Miner. Res. 1993; 8: 497-503Google Scholar). Only the latter concentrations are coupled to adenylate cyclase activity. 2) Treatment of normal keratinocytes and squamous carcinoma cell lines with N-terminal peptide fragments of both PTH and PTHRP leads to increases in intracellular Ca2+ but not cAMP (19Orloff J.J. Kats Y. Ureña P. Schipani E. Vasavada R.C. Philbrick W.M. Behal A. Abou-Samra A.-B. Segre G.V. Jüppner H. Endocrinology. 1995; 136: 3016-3023Google Scholar). The absence of a cAMP response is not a consequence of a dysfunctional signaling pathway, as squamous carcinoma cells stably transfected with the type I receptor show increased cAMP accumulation in response to PTH and PTHRP. These cells also express multiple mRNA transcripts that hybridize to type I receptor cDNA probes, yet differ significantly in size from the type I receptor mRNA present in human bone SaOS-2 cells. Differently sized transcripts are also observed in rat kidney, liver, skin, and testes (20Ureña P. Kong X.F. Abou-Samra A.-B. Jüppner H. Kronenberg H.M. Potts Jr., J.T. Segre G.V. Endocrinology. 1993; 133: 617-623Google Scholar). 3) C-terminal analogues of human PTH, such as PTH-(53–84), do not directly bind to the human PTH/PTHRP receptor type I and are unable to alter intracellular cAMP and Ca2+ levels, yet apparently retain the ability to stimulate alkaline phosphatase activity, osteoclast-like cell formation, and bone-resorbing activity by mature osteoclasts (21Murray T.M. Rao L.G. Muzaffar S.A. Ly H. Endocrinology. 1989; 124: 1097-1099Google Scholar, 22Kaji H. Sugimoto T. Kanatani M. Miyauchi A. Kimura T. Sakakibara S. Fukase M. Chihara K. Endocrinology. 1994; 134: 1897-1904Google Scholar, 23Pines M. Adams A.E. Stueckle S. Bessalle R. Rashti-Behar V. Chorev M. Rosenblatt M. Suva L.J. Endocrinology. 1994; 135: 1713-1715Google Scholar). 4) Likewise, C-terminal analogues of human PTHRP, such as PTHRP-(107–139), also do not bind to the PTH/PTHRP receptor type I, yet appear to signal by increasing intracellular Ca2+, but not cAMP, in hippocampal neurons (45Fukayama S. Tashjian Jr., A.H. Davis J.N. Chisholm J.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10182-10186Google Scholar). 5) Radioligand and affinity cross-linking studies have identified a 90-kDa protein in rat osteosarcoma (ROS 17/2.8) and rat parathyroid (PT-r3) cells that selectively binds with high affinity to the C-terminal region of PTH-(1–84) (46Inomata N. Akiyama M. Kubota N. Jüppner H. Endocrinology. 1995; 136: 4732-4740Google Scholar). 6) Apical and basolateral membranes isolated from rat renal cortical cells contain PTH/PTHRP receptors that differ quantitatively in their coupling to G-proteins and second messenger systems (47Kaufmann M. Muff R. Stieger B. Biber J. Murer H. Fischer J.A. Endocrinology. 1994; 134: 1173-1178Google Scholar). These data have been interpreted to indicate the existence of a novel PTH/PTHRP receptor(s) or an alternatively spliced variant of the type I receptor, although other explanations, such as differences in the membrane environment or the signaling repertoire of the cell, may also explain some of the data.Nevertheless, in view of the above observations, it was important to establish whether the regulation of the apical Na+/H+ exchanger NHE3 isoform by synthetic N-terminal analogues of PTH and PTHRP in renal OK cells could be solely accounted for by activation of the PTH/PTHRP receptor type I known to be expressed in this cell line. The data in this study clearly demonstrate that structurally diverse N-terminal analogues of PTH and PTHRP (i.e. PTH-(1–34), PTH-(3–34), PTH-(28–42), PTH-(28–48), and PTHRP-(1–34)) are able to directly bind to the rat PTH/PTHRP receptor type I, activate distinct second messenger systems, and elicit biological responses (i.e. inhibition of rat NHE3 activity) in a heterologous mammalian expression system.Examination of these N-terminal analogues complement and extend previous studies that have tested the effects of PTH-(1–34), PTH-(3–34), or PTH-(7–34) on second messenger production in transiently transfected COS-7 cells (9Abou-Samra A.-B. Jüppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Ureña P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Google Scholar, 37Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Google Scholar, 38Schipani E. Karga H. Karaplis A.C. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Abou-Samra A.-B. Jüppner H. Endocrinology. 1993; 132: 2157-2165Google Scholar) or stably transfected LLC-PK1 cells (4Bringhurst F.R. Jüppner H. Guo J. Ureña P. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.-B. Segre G.V. Endocrinology. 1993; 132: 2090-2098Google Scholar) expressing either the opossum, rat, or human PTH/PTHRP receptor type I. In total, these data clearly establish that the N-terminal domains of PTH and PTHRP, despite having different amino acid sequences, probably share sufficient tertiary structure to bind to the type I receptor and activate the PKA and/or PKC pathways. Whether these analogues bind to the same or different regions of the receptor is unknown. The molecular mechanism by which some of these analogues can selectively activate PKC but not PKA is an area of particular interest. Recent structural studies of the type I receptor indicate that amino acids near the N terminus (residues 31–47) and within the third extracellular loop (residues 431–440) are important for ligand-receptor interactions (48Jüppner H. Schipani E. Bringhurst F.R. McClure I. Keutmann H.T. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.-B. Segre G.V. Gardella T.J. Endocrinology. 1994; 134: 879-884Google Scholar, 49Lee C. Gardella T.J. Abou-Samra A.-B. Nussbaum S.R. Segre G.V. Potts Jr., J.T. Kronenberg H.M. Jüppner H. Endocrinology. 1994; 135: 1488-1495Google Scholar, 50Lee C.W. Luck M.D. Jüppner H. Potts Jr., J.T. Kronenberg H.M. Gardella T.J. Mol. Endocrinol. 1995; 9: 1269-1278Google Scholar), whereas the C-terminal cytoplasmic region between residues 480 and 591 influences G-proteins that regulate adenylate cyclase but not phospholipase C (51Iida-Klein A. Guo J. Xie L.Y. Jüppner H. Potts Jr., J.T. Kronenberg H.M. Bringhurst F.R. Abou-Samra A.-B. Segre G.V. J. Biol. Chem. 1995; 270: 8458-8465Google Scholar, 52Schneider H. Feyen J.H.M. Seuwen K. FEBS Lett. 1994; 351: 281-285Google Scholar).In this study, significant differences were observed in the concentration of PTH-(1–34) required to induce PTH/PTHRP receptor type I activation of adenylate cyclase (K0.5∼10−10M) and PKC (K0.5 <10−11M) activities. As only one type of receptor is present in these cells, this difference appears to be an intrinsic feature of the protein and likely reflects differential sensitivities of the two signaling pathways to fractional occupancy of a single receptor. These kinetic differences mimic that observed in the opossum renal proximal tubule OK cell line which expresses endogenous PTH/PTHRP receptor type I and apical NHE3 activity (7Azarani A. Goltzman D. Orlowski J. J. Biol. Chem. 1995; 270: 20004-20010Google Scholar, 37Jüppner H. Abou-Samra A.-B. Freeman M. Kong X.F. Schipani E. Richards J. Kolakowski Jr., L.F. Hock J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Science. 1991; 254: 1024-1026Google Scholar, 53Amemiya M. Yamaji Y. Cano A. Moe O.W. Alpern R.J. Am. J. Physiol. 1995; 269: C126-C133Google Scholar).Finally, our data indicate that the specialized apical membrane environment of OK cells is not a determining factor in the coupling of ligand-activated PTH/PTHRP receptor type I to inhibition of NHE3 activity. Although the results do not exclude the presence of other PTH/PTHRP receptors in OK and other renal proximal tubule cells, it suggests that the PTH/PTHRP receptor type I is sufficient to mediate the diverse biological actions of these N-terminal PTH and PTHRP analogues in these and possibly other cell types. INTRODUCTIONParathyroid hormone (PTH), 1The abbreviations used are: PTHparathyroid hormonePTHRPPTH-related peptidePKAprotein kinase APKCprotein kinase CDTTdithiothreitolPMSFphenylmethylsulfonyl fluoride; α-MEM. α-minimal essential mediumPMAphorbol 12-myristate 13-acetateBCECF2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein. PTH-related peptide (PTHRP), and their N-terminal analogues influence plasma calcium and phosphate homeostasis by regulating a variety of membrane ion channels and transporters within target cells, including Ca2+ channels (1Yamaguihi D.T. Hahn T.J. Iida-Klein A. Kleeman C.R. Muallem S. J. Biol. Chem. 1987; 262: 7711-7718Google Scholar), Cl− channels (2Chesnoy-Marchais D. Fritsch J. Pflügers Arch. 1989; 415: 104-114Google Scholar), Na+/Pi cotransporters (3Malmström K. Stange G. Murer H. Biochem. J. 1988; 251: 207-213Google Scholar, 4Bringhurst F.R. Jüppner H. Guo J. Ureña P. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.-B. Segre G.V. Endocrinology. 1993; 132: 2090-2098Google Scholar), and Na+/H+ exchangers (5Pollock A.S. Warnock D.G. Strewler G.J. Am. J. Physiol. 1986; 250: F217-F225Google Scholar, 6Helmle-Kolb C. Montrose M.H. Stange G. Murer H. Pflügers Arch. 1990; 415: 461-470Google Scholar, 7Azarani A. Goltzman D. Orlowski J. J. Biol. Chem. 1995; 270: 20004-20010Google Scholar, 8Azarani A. Orlowski J. Goltzman D. J. Biol. Chem. 1995; 270: 23166-23172Google Scholar). This multiplicity of actions is generally believed to reflect ligand binding to a common heterotrimeric G-protein-coupled receptor that is linked to multiple effector systems (i.e. adenylate cyclase and phospholipase C) (9Abou-Samra A.-B. Jüppner H. Force T. Freeman M.W. Kong X.F. Schipani E. Ureña P. Richards J. Bonventre J.V. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2732-2736Google Scholar). This is exemplified in renal proximal tubule OK cells where N-terminal peptide fragments of PTH and PTHRP (i.e. PTH-(1–34), PTH-(3–34), PTH-(28–42), PTH-(28–48), and PTHRP-(1–34)) rapidly inhibit the activity of the apically localized Na+/H+ exchanger NHE3 isoform by a mechanism involving cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) (7Azarani A. Goltzman D. Orlowski J. J. Biol. Chem. 1995; 270: 20004-20010Google Scholar). This regulation is remarkable given that PTH (an 84-amino acid peptide) and PTHRP (a 139–173-amino acid peptide depending on the species) (10Orloff J.J. Wu T.L. Stewart A.F. Endocr. Rev. 1989; 10: 476-495Google Scholar) share minimal identity in primary structure, with only 8 of the 13 N-terminal amino acids being common between these two peptides. PTH and PTHRP require the first two N-terminal amino acids and amino acids 25–34 to stimulate adenylate cyclase activity (11Goltzman D. Peytremann A. Callahan E. Tregear G.W. Potts Jr., J.T. J. Biol. Chem. 1975; 250: 3199-3203Google Scholar, 12Rabbani S.A. Kaiser S.M. Henderson J.E. Bernier S.M. Mouland A.J. Roy D.R. Zahab D.M. Sung W.L. Goltzman D. Hendy G.N. Biochemistry. 1990; 29: 10080-10089Google Scholar, 13Rabbani S.A. Mitchell J. Roy D.R. Hendy G.N. Goltzman D. Endocrinology. 1988; 123: 2709-2716Google Scholar, 14Rosenblatt M. N. Engl. J. Med. 1986; 315: 1004-1013Google Scholar). In contrast, amino acids 3–34 and even smaller regions (amino acids 28–34) appear sufficient to activate PKC translocation to the plasma membrane (15Fujimori A. Cheng S.L. Avioli L.V. Civitelli R. Endocrinology. 1992; 130: 29-36Google Scholar, 16Jouishomme H. Whitfield J.F. Chakravarthy B. Durkin J.P. Gagnon L. Isaacs R.J. Maclean S. Neugebauer W. Willick G. Rixon R.H. Endocrinology. 1992; 130: 53-60Google Scholar, 17Jouishomme H. Whitfield J.F. Gagnon L. Maclean S. Isaacs R. Chakravarthy B. Durkin J. Neugebauer W. Willick G. Rixon R.H. J. Bone Miner. Res. 1994; 9: 943-949Google Scholar).However, the universality of this signaling paradigm to account for the diverse actions of N-terminal analogues of PTH and PTHRP is no longer tenable in view of recent data reporting the existence of additional related receptors. Usdin et al. (18Usdin T.B. Gruber C. Bonner T.I. J. Biol. Chem. 1995; 270: 15455-15458Google Scholar) recently isolated and characterized a unique receptor (called PTH2) from a rat brain cDNA library that shares 70% amino acid identity to the classical PTH/PTHRP receptor (type I). PTH2 mRNA is expressed predominantly in brain and pancreas, and to a much lesser extent in placenta and testis. PTH2 is also functionally distinguished from the type I receptor by its selective binding of PTH and by its potent activation of adenylate cyclase activity.In addition, biochemical studies in normal keratinocytes and squamous carcinoma cell lines suggest the existence of a novel PTH/PTHRP receptor (type II) that differs qualitatively in its intracellular signaling properties from those of the type I receptor and PTH2 (19Orloff J.J. Kats Y. Ureña P. Schipani E. Vasavada R.C. Philbrick W.M. Behal A. Abou-Samra A.-B. Segre G.V. Jüppner H. Endocrinology. 1995; 136: 3016-3023Google Scholar). This potential receptor is activated by N-terminal peptide fragments of both PTH and PTHRP, leading to increases in intracellular Ca2+ but not cAMP. These cells also express multiple mRNA transcripts that hybridize to type I receptor cDNA probes, yet differ significantly in size from the type I receptor mRNA present in human bone SaOS-2 cells. Differently sized transcripts are also observed in rat kidney, liver, skin, and testes (20Ureña P. Kong X.F. Abou-Samra A.-B. Jüppner H. Kronenberg H.M. Potts Jr., J.T. Segre G.V. Endocrinology. 1993; 133: 617-623Google Scholar). These data have been interpreted to indicate the presence of a distinct gene product or an alternatively spliced variant of the type I receptor.Other circumstantial evidence also supports the existence of multiple receptors. A C-terminal peptide of human PTH (i.e. PTH-(53–84)) elicits a number of biological responses in rat osteosarcoma cells (21Murray T.M. Rao L.G. Muzaffar S.A. Ly H. Endocrinology. 1989; 124: 1097-1099Google Scholar, 22Kaji H. Sugimoto T. Kanatani M. Miyauchi A. Kimura T. Sakakibara S. Fukase M. Chihara K. Endocrinology. 1994; 134: 1897-1904Google Scholar) yet fails to bind to the human PTH/PTHRP receptor type I stably expressed in human embryonic kidney (HEK-293) cells (23Pines M. Adams A.E. Stueckle S. Bessalle R. Rashti-Behar V. Chorev M. Rosenblatt M. Suva L.J. Endocrinology. 1994; 135: 1713-1715Google Scholar). Thus, it is possible that the biological activity of some of these N- and C-terminal analogues is actually elicited by selective binding to other, as yet uncharacterized, PTH/PTHRP receptors. Understanding the signaling mechanism of these PTH/PTHRP analogues is of physiological relevance as parathyroid cells normally secrete peptide fragments of PTH (24MacGregor R.R. Jilka R.L. Hamilton J.W. J. Biol. Chem. 1986; 261: 1929-1934Google Scholar).In view of these data, we tested the hypothesis that N-terminal analogues of PTH and PTHRP can bind to and activate the PTH/PTHRP receptor type I, stimulate PKA and PKC, and acutely regulate the activity of the Na+/H+ exchanger NHE3 isoform, as is believed to occur in renal cells. This was accomplished by transient and stable transfection of cDNAs encoding the rat PTH/PTHRP receptor type I and the rat Na+/H+ exchanger NHE3 isoform into Chinese hamster ovary AP-1 cells that are devoid of endogenous Na+/H+ exchanger activity and lack responsiveness to PTH or PTHRP. The data clearly demonstrate that the AP-1-transfected cells were able to bind the various PTH and PTHRP analogues, stimulate production of multiple second messengers, and elicit biological responses in a manner that precisely mimics the responses observed in renal OK cells. Although the data do not exclude the presence of other PTH/PTHRP receptors in OK cells, it suggests that the PTH/PTHRP receptor type I is sufficient to mediate the diverse biological actions of these N-terminal PTH and PTHRP analogues in these cells.
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