Coding GNAS mutations leading to hormone resistance impair in vitro agonist- and cholera toxin-induced adenosine cyclic 3′,5′-monophosphate formation mediated by human XLαs

Most loss of function mutations of GNAS identified in different forms of pseudohypoparathyroidism disrupt not only the stimulatory G protein alpha-subunit (Gs alpha), but also its paternally expressed variant, XL alpha s. However, the possibility that XL alpha s deficiency contributes to disease pathogenesis has remained unexplored. We therefore examined the signaling property of human XL alpha s and the effects of one novel (XL alpha s(H7O4P) or Gs alpha(H362P)) and two previously described (XL alpha s(DelI724) and XL alpha s(Y733X) or Gs alpha(DelI382) and Gs alpha(Y391X), respectively) GNAS mutations on either XL alpha s or Gs alpha activity. Confocal immunofluorescence microscopy detected human XL alpha s immunoreactivity at the plasma membrane of transduced mouse embryonic fibroblasts null for endogenous Gs alpha and XL alpha s (Gnas(E2-/E2-) cells). Cholera toxin- and isoproterenol-induced cAMP accumulation in Gnas(E2-/E2-) cells transiently expressing wild-type human XL alpha s was similar to that in cells transiently expressing wild-type Gs alpha. Human XL alpha s, like Gs alpha, mediated PTH-induced cAMP accumulation in Gnas(E2-/E2-) cells coexpressing PTH receptor type 1 and either of these proteins. Moreover, overexpression of human XL alpha s or Gs alpha markedly enhanced the PTH-induced cAMP accumulation in opossum kidney cells that endogenously express PTH receptor type 1. In contrast, each XL alpha s mutant failed to mediate isoproterenol- and PTH-induced cAMP accumulation in transduced Gnas(E2-/E2-) cells. XL alpha s(DelI724) showed a reduced cholera toxin response over the basal level compared with wild-type XL alpha s, and XL alpha s(H704P) completely failed to respond to cholera toxin. These findings were comparable to those observed with each corresponding Gs alpha mutant transiently expressed in Gnas(E2-/E2-) cells. Thus, mutations that typically inactivate Gs alpha also impair XL alpha s activity, consistent with a possible role for XL alpha s deficiency in diseases caused by paternal GNAS mutations.