This project will apply a genetic approach to elucidation of the biochemical regulation of adenylate cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE), the enzymes that synthesize and degrade cyclic AMP (cAMP). We will isolate and characterize the phenotypes of vertebrate and yeast cells with mutations affecting these enzymes. Using recombinant DNA technology and transformation of mutant yeast and animal cells, we will clone the genes encoding polypeptide components of AC and PDE, and use the cloned genes to investigate the structure and regulation of the enzymes. Tools to be used include: Mutant phenotypes isolated from the S49 mouse lymphoma cell, a useful model system for selecting mammalian cells with mutations in the cAMP system; bacterial toxins, including cholera toxin, anthrax toxin, and the Islet Activating Protein of Bordetella pertussis, all of which perturb synthesis of cAMP in mammalian cells; and mutant yeast cells with defects in AC components. The overall goal of this project is to understand the molecular basis of regulation of cAMP synthesis and degradation, involving the synthesis and post-translational covalent modification of components of AC and PDE, as well as their interactions with one another in functioning cells. The health-related significance of this work derives from: 1. The pivotal importance of AC and PDE mediating or modulating the actions of a large number of hormones and drugs. To the extent that molecular regulation of these enzymes is understood, pharmacotherapy can be made more specific and effective. 2. An inherited human disease, pseudohypoparathyroidism, is caused by a mutation affecting hormone-sensitive AC; better understanding of the genetics and molecular regulation of AC will help in understanding this disease, and therefore in treating and preventing it.
