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A Novel Mutation Adjacent to the Switch III Domain of G_s in a Patient with Pseudohypoparathyroidism

Membrane Biochemistry Section (D.R.W.), Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, Clinical Neurogenetics Branch (P.V.G.), National Institute of Mental Health, Metabolic Diseases Branch (R.M.C., L.S.W.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892

A novel G_s mutation encoding the substitution of arginine for serine 250 (G_s S250R) was identified in a patient with pseudohypoparathyroidism type Ia. Both G_s activity and G_s expression were decreased by about 50% in erythrocyte membranes from the affected patient. The cDNA of this G_s mutant, as well as one in which the S250 residue is deleted (G_s-S250), was generated, and the biochemical properties of the products of in vitro transcription/translation were examined. Both mutants had a sedimentation coefficient similar to that of wild type G_s (3.7S) when kept at 0 C after synthesis. However when maintained for 1–2 h at 30–37 C, both mutants aggregated to a material sedimenting at 6.3S or greater (G_s-S250R to a greater extent than G_s-S250), while wild type G_s sedimented at 3.7S, suggesting that the mutants were thermolabile. Incubation in the presence of high doses of guanine nucleotide partially prevented heat denaturation of G_s S250 but had no protective effect on G_s-S250R. Sucrose density gradient centrifugation at 0 C in the presence and absence of ß-dimers demonstrated that, in contrast to wild type G_s, neither mutant could interact with ß. Trypsin protection assays revealed no protection of G_s-S250R by GTPS or AlF₄^- at any temperature. GTPS conferred modest protection of G_s-S250 (50% of wild-type G_s) at 30 C but none at 37 C, while AlF₄^- conferred slight protection at 20 C but none at 30 C or above. Consistent with this result, G_s-S250 was able to stimulate adenylyl cyclase at 30 C when reconstituted with cyc^- membranes in the presence of GTPS but not in the presence of AlF₄^-. G_s-S250R showed no ability to stimulate adenylyl cyclase in the presence of either agent. Stable transfection of mutant and wild-type G_s into cyc^- S49 lymphoma cells revealed that the majority of wild type G_s localized to membranes, while little or no membrane localization occurred for either mutant. Modeling of G_s based upon the crystal structure of G_t or G_i suggests that Ser²⁵⁰ interacts with several residues within and around the conserved NKXD motif, which directly interacts with the guanine ring of bound GDP or GTP. It is therefore possible that substitution or deletion of this residue may alter guanine nucleotide binding, which could lead to thermolability and impaired function.

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