The N-terminal region of the third intracellular loop of the parathyroid hormone (PTH)/PTH-related peptide receptor is critical for coupling to cAMP and inositol phosphate/Ca2+ signal transduction pathways.
Structural determinants within the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor that mediate G-protein activation of adenylate cyclase and phospholipase C are unknown. We investigated the role of the N-terminal region of the third intracellular loop of the opossum PTH/PTHrP receptor in coupling to two signal transduction pathways. We mutated residues in this region by tandem-alanine scanning and expressed these mutant receptors in COS-7 cells and/or Xenopus oocytes. All mutant receptors retained high affinity PTH binding in COS-7 cells, indistinguishable from wild-type receptors. Receptors with tandem-alanine substitutions in two N-terminal segments (377RVL379 and 381TKLR384) demonstrated impaired adenylate cyclase and phospholipase C activation. Receptor mutants with single-alanine substitutions scanning these two segments showed three different signaling defects in COS-7 cells. 1) Two mutant receptors (V378A and L379A) had reduced inositol phosphate (IP), but normal cAMP responses to PTH. 2) Mutant receptor T381A showed reduced cAMP, but wild-type IP responses to PTH. 3) Mutant receptor K382A demonstrated both markedly reduced cAMP and IP production due to PTH. In oocytes, mutants T381A and K382A showed decreased PTH-stimulated cAMP accumulation and intracellular Ca2+ mobilization. Thus, the N-terminal region of the third intracellular loop of this receptor plays a critical role in coupling to both Gs- and Gq-mediated second-messenger generation.
Huang
Z
Z
Endocrine Unit, Department of Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, California 94121, USA.
Chen
Y
Y
Pratt
S
S
Chen
T H
TH
Bambino
T
T
Nissenson
R A
RA
Shoback
D M
DM
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D005260:Female; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009865:Oocytes / Q000378:metabolism; D010281:Parathyroid Hormone / Q000378:metabolism; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D015398:Signal Transduction; D014981:Xenopus
D007295:Inositol Phosphates; D010281:Parathyroid Hormone; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D000242:Cyclic AMP; D002118:Calcium