A novel mutation adjacent to the switch III domain of G(S alpha) in a patient with pseudohypoparathyroidism.
A novel G(S alpha) mutation encoding the substitution of arginine for serine 250 (G[S alpha] S250R) was identified in a patient with pseudohypoparathyroidism type Ia. Both G(S) activity and G(S alpha) expression were decreased by about 50% in erythrocyte membranes from the affected patient. The cDNA of this G(S alpha) mutant, as well as one in which the S250 residue is deleted (G[S alpha]-deltaS250), 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 alpha) (approximately 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 approximately 6.3S or greater (G[S alpha]-S250R to a greater extent than G(S alpha]-deltaS250), while wild type G(S alpha) sedimented at approximately 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 alpha) deltaS250 but had no protective effect on G(S alpha-S250R. Sucrose density gradient centrifugation at 0 C in the presence and absence of beta gamma-dimers demonstrated that, in contrast to wild type G(S alpha) neither mutant could interact with beta gamma. Trypsin protection assays revealed no protection of G(S alpha)-S250R by GTPgammaS or AIF4- at any temperature. GTPgammaS conferred modest protection of G(S alpha)-deltaS250 (approximately 50% of wild-type G[S alpha]) at 30 C but none at 37 C, while AIF4- conferred slight protection at 20 C but none at 30 C or above. Consistent with this result, G(S alpha)-deltaS250 was able to stimulate adenylyl cyclase at 30 C when reconstituted with cyc- membranes in the presence of GTPgammaS but not in the presence of AIF4-. G(S alpha)-S250R showed no ability to stimulate adenylyl cyclase in the presence of either agent. Stable transfection of mutant and wild-type G(S alpha) into cyc- S49 lymphoma cells revealed that the majority of wild type G(S alpha) localized to membranes, while little or no membrane localization occurred for either mutant. Modeling of G(S alpha) based upon the crystal structure of G(t alpha) or G(i alpha) 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.
Warner
D R
DR
Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Gejman
P V
PV
Collins
R M
RM
Weinstein
L S
LS
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D004910:Erythrocyte Membrane / Q000378:metabolism; D005260:Female; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000235:genetics*; D005812:Genes, Switch; D006579:Heterozygote; D006801:Humans; D008223:Lymphoma / Q000473:pathology; D051379:Mice; D008875:Middle Aged; D008958:Models, Molecular; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011547:Pseudohypoparathyroidism / Q000235:genetics*; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D019205:GTP-Binding Protein alpha Subunits, Gs