guanosine-5--o-(3-thiotriphosphate) and Pseudohypoparathyroidism

Luteinizing hormone stimulates testicular Leydig cells to produce testosterone by binding to a receptor that activates the G protein Gs and adenylyl cyclase. Testotoxicosis is a form of precocious puberty in which the Leydig cells secrete testosterone in the absence of luteinizing hormone, often due to constitutive activation of the luteinizing hormone receptor and (indirectly) Gs (refs 1-4). Here we study two unrelated boys suffering from a paradoxical combination of testotoxicosis and pseudohypoparathyroidism type Ia (PHP-Ia), a condition marked by resistance to hormones acting through cyclic AMP (parathyroid hormone and thyroid-stimulating hormone) as well as a 50% decrease in erythrocyte Gs activity (the remaining 50% is due to the normal Gs allele). In both patients, a mutation in the gene encoding the Gs alpha-subunit replace alanine at position 366 with serine. We show that this alpha s-A366S mutation constitutively activates adenylyl cyclase in vitro, causing hormone-independent cAMP accumulation when expressed in cultured cells, and accounting for the testotoxicosis phenotype (as cAMP stimulates testosterone secretion). Although alpha s-A366S is quite stable at testis temperature, it is rapidly degraded at 37 degrees C explaining the PHP-Ia phenotype caused by loss of Gs activity. In vitro experiments indicate that accelerated release of GDP causes both the constitutive activity and the thermolability of alpha s-A366S.

Topics: Adenylyl Cyclases; Animals; Body Temperature; Cell Line; Cyclic AMP; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Leydig Cells; Male; Point Mutation; Pseudohypoparathyroidism; Recombinant Proteins; Testicular Diseases; Transfection

Hormone-sensitive adenylate cyclase contains a recently discovered protein component that is required for stimulation of cyclic AMP synthesis by hormones and guanine nucleotides. We measured this protein in erythrocyte membranes of ten patients with pseudohypoparathyroidism (PHP), using assays of its biochemical activity and of its susceptibility to radiolabeling in the presence of 32P-NAD and cholera toxin. By both assays, the protein was reduced by 50% in erythrocytes of 4 PHP patients, as compared with normal and hypoparathyroid subjects. These 4 subjects, in contrast to the 6 PHP patients (5 in one family) whose erythrocytes contained apparently normal amounts of the cyclase component, exhibited the full spectrum of skeletal abnormalities found in PHP. We conclude that partial deficiency of the guanine nucleotide regulatory protein is a biochemical marker for a subset of PHP patients. If present in other tissues, this deficiency could explain the resistance of target organs in PHP to parathormone and other hormones that work via cyclic AMP.

Topics: Adenylyl Cyclases; Cholera Toxin; Cyclic AMP; Erythrocyte Membrane; Erythrocytes; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Isoproterenol; Mutation; Pseudohypoparathyroidism; Thionucleotides