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. AU 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 ((377)RVL(379) and (381)TKLR(384)) 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 G(s)- and G(q)-mediated second-messenger generation.