TRUNCATION AND ALANINE-SCANNING MUTANTS OF TYPE-I ADENYLYL-CYCLASE

A variety of truncated constructs of type I and type II adenylyl cyclase have been synthesized in Sf9 cells using recombinant baculoviruses, as have a number of type I adenylyl cyclases with point mutations. Truncations indicate that the nonconserved C-1b and C-2b domains of adenylyl cyclase are not necessary for regulation of enzymatic activity by G(s alpha) and forskolin. Point mutations demonstrate the requirement for both of the conserved (and homologous) domains of adenylyl cyclase (C-1a and C-2a) and the nonequivalence of these domains. Linkage of certain effects of mutations on the K-m for substrate with alterations of the characteristics of beta-site inhibition suggest that ATP and beta-site inhibitors may bind to different conformations of the same site. However, other mutations affected only alpha-site inhibition. Although the mutations studied have not permitted assignment of unique functions to the two homologous domains, they have revealed novel phenotypes that appear to reflect the regulatory complexity of mammalian membrane-bound adenylyl cyclases, including the possibility of oligomerization of the enzymes.