Hormone-sensitive adenylyl cyclase is a model system for the study of receptor-mediated signal transduction. It is compromised of three types of components: 1.) receptors for hormones that regulate cyclic AMP (cAMP) synthesis, 2.) regulatory GTP binding proteins (G proteins), and 3.) the family of enzymes, the adenylyl cyclases. Hormone binding to its receptor results in modulation of the enzyme's ability to catalyze the Mg2+- dependent formation of cAMP from ATP at the intracellular face of the plasma membrane. Concentrations of cAMP are altered by at least 35 different stimulatory or inhibitory hormones and neurotransmitters. The second messenger, cAMP, propagates the hormone signal by combining with cAMP-dependent protein kinase. Physiological effects such as the control of glycogenolysis and heart rate are, at least in part, regulated by this pathway. In addition, other signalling pathways may influence the cAMP pathway by regulation of particular adenylyl cyclase subtypes through the effects of Ca2+/calmodulin, protein kinase C, or other kinases. Despite its central role in the cAMP signalling pathway, adenylyl cyclases have resisted the extensive biochemical scrutiny they merit because of their instability and low abundance. Recently a cDNA clone for a calmodulin-sensitive form of bovine brain adenylyl cyclase was isolated. This has made it possible to clone cDNAs for three other forms of the enzyme. The structural diversity within this family of enzymes will be examined by molecular cloning techniques. Probes based on the sequences of the four known adenylyl cyclases will be utilized in the analysis of RNA blots to assess the extent of the diversity in the adenylyl cyclase family. These same probes will also facilitate the isolation of cDNAs encoding other forms of adenylyl cyclase. Both oligonucleotide and peptide-specific antibody probes derived from the sequence of novel clones will be utilized to determine in which tissues a particular subtype of the enzyme is expressed. Full-length cDNAs will be expressed in mammalian cell systems to prove they express functional adenylyl cyclases, and to examine their interactions with other cellular components. Substantial quantities of recombinant adenylyl cyclases will be purified from membranes of insect cells infected with appropriate baculovirus constructs. The response of the recombinant protein to a variety of regulators will be characterized in both systems. The function of various domains within the adenylyl cyclase structure will be assessed by the construction of mutants, and the analysis of their phenotypes following expression in both mammalian cells and baculovirus-infected cells.
