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 C1b and C2b domains of adenylyl cyclase are not necessary for regulation of enzymatic activity by Gs alpha and forskolin. Point mutations demonstrate the requirement for both of the conserved (and homologous) domains of adenylyl cyclase (C1a and C2a) and the nonequivalence of these domains. Linkage of certain effects of mutations on the Km for substrate with alterations of the characteristics of P-site inhibition suggest that ATP and P-site inhibitors may bind to different conformations of the same site. However, other mutations affected only P-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.
Tang
W J
WJ
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637, USA.
Stanzel
M
M
Gilman
A G
AG
D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D000262:Adenylyl Cyclases; D000409:Alanine