Pseudohypoparathyroidism type Ia (PHP-Ia) results from the loss of one allele of G s␣, causing resistance to parathyroid hormone and other hormones that transduce signals via G s. Most Gs␣mutations cause the complete loss of protein expression, but some cause loss of function only, and these have provided valuable insights into the normal function of G proteins. Here we have analyzed a mutant G s␣ (␣s-AVDT) harboring AVDT amino acid repeats within its GDP/GTP binding site, which was identified in unique patients with PHP-Ia accompanied by neonatal diarrhea. Biochemical and intact cell analyses showed that ␣s-AVDT is unstable but constitutively active as a result of rapid GDP release and reduced GTP hydrolysis. This instability underlies the PHP-Ia phenotype. ␣s-AVDT is predominantly localized in the cytosol, but in rat and mouse small intestine epithelial cells (IEC-6 and DIF-12 cells) ␣s-AVDT was found to be localized predominantly in the membrane where adenylyl cyclase is present and constitutive increases in cAMP accumulation occur in parallel. The likely cause of this membrane localization is the inhibition of an activation-dependent decrease in ␣s palmitoylation. Upon the overexpression of acyl-protein thioesterase 1, however, ␣s-AVDT translocates from the membrane to the cytosol, and the constitutive accumulation of cAMP becomes attenuated. These results suggest that PHP-Ia results from the instability of ␣s-AVDT and that the accompanying neonatal diarrhea may result from its enhanced constitutive activity in the intestine. Hence, palmitoylation may control the activity and localization of G s␣ in a cell-specific manner.disease ͉ G protein ͉ lipid modification ͉ localization ͉ activity G protein diseases have revealed key pathways underlying physiologic regulation and the molecular mechanisms involved. Pseudohypoparathyroidism type Ia (PHP-Ia) is a classic example of such a disease and results from the heterozygous loss of function of G s␣ (1-4). In most cases, the loss of G s␣ function results from a loss of protein expression due to insertions, deletions, frameshift deletions, nonsense mutations, and splice junction mutations. In some cases, however, PHP-Ia occurs in the presence of G s␣ protein expression, and analyses of the corresponding mutations have greatly furthered our understanding of how G proteins function normally (5).The activity of G s␣ is cyclically regulated via two unidirectional steps, a GDP/GTP exchange and the hydrolysis of GTP (5-8). The agonist-occupied receptor accelerates GDP release from the ␣␥ trimer, which allows GTP to bind to the empty guanine nucleotide pocket of G ␣ and thus induce a conformational change that enables its dissociation from the receptor and ␥ subunit. The GTP-bound G ␣ and ␥ dimer transmit a signal until GTP is hydrolyzed, which then allows the GDP-bound G ␣ to bind and inactivate G ␥ .The defects resulting from G s␣ mutations that do not prevent protein expression but nevertheless result in the onset of PHP-Ia have now been elucidated at the molecular l...