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 [I-125-Tyr(36)]PTHRP-(1-36)-NH2 radio-ligand 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-(334), 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.