Pseudohypoparathyroidism, a Novel Mutation in the βγ-Contact Region of Gsα 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 ␣ subunit (␣ 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 ␣ s . We assessed signaling function of ␣ s -WT versus ␣ s -R231H transiently transfected in HEK293 cells. Hormone receptor-dependent stimulation of cAMP accumulation in cells expressing ␣ s -R231H is reduced by ϳ75% in comparison to cAMP accumulation in cells expressing ␣ s -WT. A second mutation, ␣ s -R201C, inhibits the GTPase turnoff reaction of ␣ s , thus producing receptor-independent stimulation of cAMP accumulation. The double mutant, ␣ s -R231H/ R201C, stimulates cAMP accumulation almost as well (ϳ80%) as does ␣ 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 ␤␥ subunit rather than with the hormone receptor. Thus, the R231H phenotype suggests that switch 2 (perhaps in concert with ␤␥) mediates G protein activation by receptors at a site distant from the receptor-G protein contact surface.Heterotrimeric G proteins relay information from receptors for extracellular stimuli to effector enzymes and ion channels that mediate cell responses. Receptors interact with the G protein ␣/␤␥ complex, causing release of GDP bound in the guanine nucleotide binding pocket of the G protein ␣ subunit and its replacement by GTP. GTP-induced conformational changes cause G␣GTP to dissociate from the ␤␥ dimer, allowing both subunits to interact with effectors. Effector stimulation is then terminated by the intrinsic GTPase activity of G␣, followed by reassociation of G␣GDP and ␤␥. With respect to this cycle, recently published crystal structures have revealed details of GTP-induced conformational change in G␣ (1-3), a plausible structure for the catalytic intermediate in the GTPase reaction (4, 5), and three-dimensional structures for the G␣␤␥ heterotrimer (6, 7). Because we do not have the structure of a receptor-G␣␤␥ complex, however, we cannot yet describe in molecular detail the pivotal event in receptor-G protein signaling, receptor-triggered release of GDP from G␣.At present we can try to understand this event by interpreting effects of instructive mutations. A potential source of such mutations is an inherited disease of G protein signaling, pseudohypoparathyroidism, type Ia (PHP-Ia) (8). PHP-Ia patients inherit a defect in one of the two autosomal alleles of the gene for ␣ s , the ␣ subunit of G s , the stimulatory regulator of adenylyl cyclase. Although G s mediates effects of many hormones, the ϳ50% decrease in G s activity produced by loss of an autosomal allele cau...

Two mutations of the Gsα gene in two Japanese patients with sporadic pseudohypoparathyroidism type Ia

“…According to their in-vitro study, this mutant Gs protein impaired the responsiveness of Gsα to receptor stimulation, leading to PTH resistance. These findings of Farfel et al (1996) confirm that the disease in patient 2 is caused by the R231H mutation.…”

“…Therefore, this mutation may be the cause of the disease. Concerning the R231H mutation, Farfel et al (1996) reported the identical mutation in three patients with PHP-Ia. According to their in-vitro study, this mutant Gs protein impaired the responsiveness of Gsα to receptor stimulation, leading to PTH resistance.…”

“…The R231H mutation was reported previously . Data for mutations were derived from the following references (Ahmed et al 1998;Farfel et al 1996;Fischer et al 1998;Iiri et al 1994;Miric et al 1993;Oude-Luttikhuis et al 1994;Pattern et al 1990;Schwindinger et al 1994;Shapira et al 1996;Warner et al 1997Warner et al , 1998Weinstein et al 1990Weinstein et al , 1992Yu et al 1995Yu et al , 1999 NA, Not available; PTH, parathyroid hormone; TRH, thyroidstimulating hormone (TSH) releasing hormone; T4, thyroxine; T3, triiodothyronine a PTH infusion test was performed using bolus synthetic PTH-(1-34), at 100 U/m 2 . Basal levels are the mean urinary cyclic (c)AMP (nmol/h) and urinary phosphate-to-creatine ratio for two urine samples taken in the 2 h before the administration of PTH.…”

“…1) (Ahmed et al 1998;Farfel et al 1996;Fischer et al 1998;Iiri et al 1994;Miric et al 1993;OudeLuttikhuis et al 1994;Pattern et al 1990;Schwindinger et al 1994;Shapira et al 1996;Warner et al 1997Warner et al , 1998Weinstein et al 1990Weinstein et al , 1992Yu et al 1995Yu et al , 1999. The widespread deficiency of Gs protein can explain the resistance to PTH and other hormones whose receptors are coupled to Gs; however, the relationship between AHO phenotypes and Gsα mutations remains unclear.…”

Human G sα mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle

“…The ␣s-A366S defect shows not only a loss of function resulting from rapid denaturation of the G s␣ protein but also a gain of function resulting from its relative stabilization at 33°C (12). Additional loss-of-function G s␣ mutations include ␣s-R231H, which confers a conditionally defective activation (13,14), and ␣s-R280K, which may be defective in G proteineffector interactions (10). Moreover, some missense G s␣ mutants (S250R and R258W), which are not expressed in human cells but can be generated by in vitro translation, have also contributed to our understanding of the kinetics of GTPase in the G ␣ cycle (15,16).…”