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.
Azarani
A
A
Department of Physiology, McGill University and the Calcium Research Laboratory, Royal Victoria Hospital, Montreal H3A 1Y6, Canada.
Goltzman
D
D
Orlowski
J
J
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D010281:Parathyroid Hormone / Q000378:metabolism / Q000494:pharmacology*; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D018016:Receptors, Parathyroid Hormone / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014162:Transfection
D004791:Enzyme Inhibitors; D007546:Isoquinolines; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011506:Proteins; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; C000613385:SLC9A3 protein, human; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C