The action of many hormones requires their interaction with specific cell surface receptors that are coupled by the guanine nucleotide- binding protein G, to stimulation of adenylyl cyclase. Transmembrane signal transduction can be impaired as a result of synthesis of receptors or hormones with altered biological activity. In addition, altered hormone action can occur as a consequence of disordered G, function. This protocol examines the molecular basis for altered signal transduction in patients with disordered G protein function (e.g pseudohypoparathyroidism type la, McCune Albright syndrome, and familial alcoholism), patients with abnormal G protein-coupled receptor function (eg. impaired activation of the receptor for parathyroid hormone (PTH) in pseudohypoparathyroidism type lb), and patients with abnormal hormone function (eg Familial Isolated Hypoparathyroidism). Biochemical and clinical characterization of the patients phenotype facilitates design of laboratory studies that are used to evaluate hypotheses regarding molecular mechanisms of disease. Over the past year we have focused primarily on elucidation of the molecular basis for PTH resistance in subjects with pseudohypoparathyroidism (PHP) type lb, an autosomal syndrome in which hormone resistance is limited to PTH target tissues and associated with normal G protein activity. We have examined and characterized the biochemical phenotype of subjects with PTH resistance and their unaffected relatives from multiplex kindreds. Peripheral blood lymphoblasts were transformed with Epstein-Barr virus, and genomic DNA and total RNA were isolated from cultures of the transformed B lymphocytes. We used the polymerase chain reaction to amplify PTH receptor gene and CDNA sequences 25 patients with PHP type lb. We found neutral polymorphisms in several patients, but were unable to detect any mutations in the coding region of the receptor that could account for decreased receptor activity. We then used polymorphisms in the PTH/PTHrP and Ga, gene to perform linkage analysis in members of four large multiplex PHP type lb kindreds. All three kindreds were sufficiently informative with these markers that we were able to exclude linkage of PTH resistance to the PTH/PTHrP receptor gene. In two families linkage to the Gas, gene was similarly excluded, but in one family we found linkage to a Gas, gene allele. Analysis of the Gas, gene disclosed a three-base deletion in axon 13 in all four affected subjects from this family with PTH resistance. This mutation results in the in-frame deletion of isoleucine at codon 382 (AI382), which is in a region that appears important for receptor interaction. The three sons who carry this mutation have PTH resistance whereas their mother shows normal responsiveness to PTH. In spite of the defect in Gas, hormone resistance appears to be limited to PTH target tissues.
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