These studies are directed toward the regulatory protein (Gs) of adenylate cyclase. Adenylate cyclase is composed of at least three protein components: 1) hormone receptors, 2) a catalytic moiety, and 3) a regulatory protein (Gs) that binds guanine nucleotides. Gs functions to modulate agonist-occupied receptor affinity, and to couple hormone-receptor complexes to the catalytic unit. Gs thus regulates the hormonal activation of adenylate cyclase. Strong evidence supports a defect in adenylate cyclase as the cause of hormone resistance in pseudohypoparathyroidism (PHP), a metabolic disorder characterized by resistance of the target organs bone and kidney to the actions of parathyroid hormone. Recently, a deficiency in Gs activity has been described in cell membranes from many subjects with PHP. Unlike tissue-specific hormone receptors, Gs is similar in all cells, and deficient Gs activity is implicated as the cause of decreased synthesis of cAMP and hormone resistance in diverse tissues in many patients with PHP. For these reasons it is both interesting and important to study further the structure and mechanism(s) of action of Gs. These studies propose 1) to purify Gs from human erythrocyte membranes by a novel approach: cholera toxin catalyzes the covalent ADP-ribosylation of a 42,000-dalton subunit of Gs; T4 RNA ligase can attach polyadenylate specifically to the ADP-ribosylated Gs peptide and oligo- d(T) affinity chromatography will be used to resolve the modified Gs from other membrane proteins. 2) to use the purified (modified) Gs peptide in the production of reagent antibodies to Gs; 3) to use these antibodies in the development and application of methods for assessing the immunologic and biologic properties of Gs from erythrocyte membranes of subjects with PHP; 4) to determine the applicability of the oligo d(T) chromatography methods to the resolution of other proteins (e.g., transducin, and the inhibitory guanine nucleotide binding protein) that are specific ADP-ribose substrates. These studies may help to elucidate molecular lesions(s) in Gs which impair adenylate cyclase hormone responsiveness, and may provide further insight into hormone resistance in PHP. Ultimately, it is hoped that an immunologic approach to the study of the mechanism(s) of action of Gs will help identify the domain or subunits of Gs that regulate the complex interactions of the regulatory protein with hormone receptors and the catalytic unit.
| Murray, T M; Rao, L G; Wong, M M et al. (1993) Pseudohypoparathyroidism with osteitis fibrosa cystica: direct demonstration of skeletal responsiveness to parathyroid hormone in cells cultured from bone. J Bone Miner Res 8:83-91 |
| Ahn, T G; Antonarakis, S E; Kronenberg, H M et al. (1986) Familial isolated hypoparathyroidism: a molecular genetic analysis of 8 families with 23 affected persons. Medicine (Baltimore) 65:73-81 |
