A novel G_sα mutation encoding the substitution of arginine for serine 250 (G_sα S250R) was identified in a patient with pseudohypoparathyroidism type Ia. Both G_s activity and G_sα expression were decreased by about 50% in erythrocyte membranes from the affected patient. The cDNA of this G_sα mutant, as well as one in which the S250 residue is deleted (G_sα-ΔS250), 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_sα (∼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_sα-S250R to a greater extent than G_sα-ΔS250), while wild type G_sα 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_sα ΔS250 but had no protective effect on G_sα-S250R. Sucrose density gradient centrifugation at 0 C in the presence and absence of βγ-dimers demonstrated that, in contrast to wild type G_sα, neither mutant could interact withβγ . Trypsin protection assays revealed no protection of G_sα-S250R by GTPγS or AlF₄⁻ at any temperature. GTPγS conferred modest protection of G_sα-ΔS250 (∼50% of wild-type G_sα) 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_sα-ΔS250 was able to stimulate adenylyl cyclase at 30 C when reconstituted with cyc⁻ membranes in the presence of GTPγS but not in the presence of AlF₄⁻. G_sα-S250R showed no ability to stimulate adenylyl cyclase in the presence of either agent. Stable transfection of mutant and wild-type G_sα into cyc⁻ S49 lymphoma cells revealed that the majority of wild type G_sα localized to membranes, while little or no membrane localization occurred for either mutant. Modeling of G_sα based upon the crystal structure of G_tα or G_iα 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.