Pseudohypoparathyroidism, a novel mutation in the βγ-contact region of G(s)α impairs receptor stimulation

Abstract

Pseudohypoparathyroidism, type Ia (PHP-Ia), is a dominantly inherited endocrine disorder characterized by resistance to hormones that act by stimulating adenylyl cyclase. It is caused by inheritance of an autosomal mutation that inactivates the a subunit (α(s)) of G(s), the stimulatory regulator of adenylyl cyclase. In three members of a family, the PHP-Ia phenotype is associated with a mutation (R231H) that substitutes histidine for an arginine at position 231 in a(s). We assessed signaling function of α(s)- WT versus α(s)-R231H transiently transfected in HEK293 cells. Hormone receptor- dependent stimulation of cAMP accumulation in cells expressing α(s)-R231H is reduced by ~75% in comparison to cAMP accumulation in cells expressing α(s)- WT. A second mutation, α(s)-R201C, inhibits the GTPase turnoff reaction of α(s), thus producing receptor-independent stimulation of cAMP accumulation. The double mutant, α(s)-R231H/R201C, stimulates cAMP accumulation almost as well (~80%) as does α(s)-R201C itself, indicating that the R231H mutation selectively impairs receptor-dependent signaling. In three-dimensional structures of G protein heterotrimers, Arg-231 is located in a region, switch 2, that is thought to interact with the βγ subunit rather than with the hormone receptor. Thus, the R231H phenotype suggests that switch 2 (perhaps in concert with βγ) mediates G protein activation by receptors at a site distant from the receptor-G protein contact surface.