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

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. 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 the activity of the Na+/H+ exchanger NHE3 as is believed to in renal cells. This was by and of cDNAs encoding the rat PTH/PTHRP receptor type I and the rat Na+/H+ exchanger NHE3 isoform into Chinese hamster ovary cells that are of endogenous Na+/H+ exchanger activity and lack to PTH or data that the cells were to bind the PTH and PTHRP stimulate of multiple and elicit biological responses in a that the responses observed in renal OK cells. the data not the presence of PTH/PTHRP in OK cells, it that the PTH/PTHRP receptor type I is sufficient to the diverse biological actions of these N-terminal PTH and PTHRP analogues in these PTH/PTHRP receptor type I been from 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, H. Abou-Samra A.-B. Freeman M. Kong X.F. Schipani E. Richards J. Jr., J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Scholar, E. H. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Abou-Samra A.-B. Jüppner H. Endocrinology. 1993; 132: Scholar, J. D. N. M. J. G. C. Goltzman D. Hendy G.N. 1994; Scholar) and to a of G-protein-coupled including for peptide and adenylate peptide G.V. 1993; Scholar, T.B. E. Bonner T.I. Endocrinology. 1993; 133: Scholar, R. K. M. W. Proc. Natl. Acad. Sci. U. S. A. 1993; Scholar, D. C. C. J. L. 1993; Scholar). of these membrane (i.e. and the ability to activate that the adenylate and/or phospholipase the ability of a PTH/PTHRP receptor (i.e. type I) to to multiple effector systems was generally believed to account for the effects of PTH and PTHRP and their analogues in target (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 the of a that is expressed in brain and 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 lines of also indicate that additional PTH/PTHRP N-terminal analogues of PTH and PTHRP activate PKC activity in osteosarcoma cells in a with of activity and the 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, L. H. Whitfield J.F. Durkin J.P. Maclean S. Neugebauer W. Willick G. Rixon R.H. Chakravarthy B. J. Bone Miner. Res. 1993; Scholar). the are to adenylate cyclase activity. 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 (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). of a response is not a of a signaling as squamous carcinoma cells stably transfected with the type I receptor in response to PTH and 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). C-terminal analogues of human as not directly bind to the human PTH/PTHRP receptor type I and are unable to intracellular and Ca2+ yet the ability to stimulate cell and activity by (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, M. Adams A.E. Stueckle S. Bessalle R. Rashti-Behar V. Chorev M. Rosenblatt M. Suva L.J. Endocrinology. 1994; 135: 1713-1715Google Scholar). Likewise, C-terminal analogues of human PTHRP, as also not bind to the PTH/PTHRP receptor type I, yet appear to by intracellular but not in S. Jr., Proc. Natl. Acad. Sci. U. S. A. 1995; Scholar). and studies have a protein in rat osteosarcoma and rat parathyroid cells that selectively with to the C-terminal of N. M. N. Jüppner H. Endocrinology. 1995; 136: Scholar). and isolated from rat renal cells PTH/PTHRP that differ in their to and systems M. R. B. J. Murer H. Endocrinology. 1994; 134: Scholar). These data have been interpreted to indicate the existence of a novel PTH/PTHRP or an alternatively spliced variant of the type I as in the membrane or the signaling of the also some of the in view of the it was to the regulation of the Na+/H+ exchanger NHE3 isoform by N-terminal analogues of PTH and PTHRP in renal OK cells be for by activation of the PTH/PTHRP receptor type I to be expressed in this cell data in this that 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 to directly bind to the rat PTH/PTHRP receptor type I, activate distinct and elicit biological responses (i.e. inhibition of rat NHE3 in a expression of these N-terminal analogues and studies that have tested the effects of PTH-(1–34), PTH-(3–34), or on in transfected 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, H. Abou-Samra A.-B. Freeman M. Kong X.F. Schipani E. Richards J. Jr., J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Scholar, E. H. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Abou-Samra A.-B. Jüppner H. Endocrinology. 1993; 132: Scholar) or stably transfected cells 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) either the or human PTH/PTHRP receptor type In these data that the N-terminal of PTH and PTHRP, amino acid share sufficient to bind to the type I receptor and activate the PKA and/or PKC these analogues bind to the or regions of the receptor is mechanism by some of these analogues can selectively activate PKC but not PKA is an of studies of the type I receptor indicate that amino acids the and within the are for H. Schipani E. F.R. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.-B. Segre G.V. T.J. Endocrinology. 1994; 134: Scholar, C. T.J. Abou-Samra A.-B. Segre G.V. Potts Jr., J.T. Kronenberg H.M. Jüppner H. Endocrinology. 1994; 135: Scholar, Jüppner H. Potts Jr., J.T. Kronenberg H.M. T.J. 1995; 9: Scholar), whereas the C-terminal between and that adenylate cyclase but not phospholipase A. Guo J. Jüppner H. Potts Jr., J.T. Kronenberg H.M. F.R. Abou-Samra A.-B. Segre G.V. J. Biol. Chem. 1995; 270: Scholar, H. K. 1994; Scholar).In this were observed in the of PTH-(1–34) to PTH/PTHRP receptor type I activation of adenylate cyclase and PKC only type of receptor is present in these cells, this to be an of the protein and of the two signaling to of a receptor. These that observed in the renal proximal tubule OK cell endogenous PTH/PTHRP receptor type I and NHE3 activity (7Azarani A. Goltzman D. Orlowski J. J. Biol. Chem. 1995; 270: 20004-20010Google Scholar, H. Abou-Samra A.-B. Freeman M. Kong X.F. Schipani E. Richards J. Jr., J. Potts Jr., J.T. Kronenberg H.M. Segre G.V. Scholar, M. Y. A. R.J. Am. J. Physiol. 1995; data indicate that the membrane of OK cells is not a in the of PTH/PTHRP receptor type I to inhibition of NHE3 activity. the not the presence of PTH/PTHRP in OK and renal proximal tubule cells, it that the PTH/PTHRP receptor type I is sufficient to the diverse biological actions of these N-terminal PTH and PTHRP analogues in these and cell 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 the activity of the Na+/H+ exchanger NHE3 as is believed to in renal cells. This was by and of cDNAs encoding the rat PTH/PTHRP receptor type I and the rat Na+/H+ exchanger NHE3 isoform into Chinese hamster ovary cells that are of endogenous Na+/H+ exchanger activity and lack to PTH or data that the cells were to bind the PTH and PTHRP stimulate of multiple and elicit biological responses in a that the responses observed in renal OK cells. the data not the presence of PTH/PTHRP in OK cells, it that the PTH/PTHRP receptor type I is sufficient to the diverse biological actions of these N-terminal PTH and PTHRP analogues in these cells.