Isolation of Inactive and G Protein-resistant Adenylyl Cyclase Mutants Using Random Mutagenesis

doi:10.1074/jbc.270.39.22693

Isolation of Inactive and G Protein-resistant Adenylyl Cyclase Mutants Using Random Mutagenesis (*)

From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

^¶ To whom all correspondence should be addressed:
Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore MD 21205.
Tel.: 410-955-4699; Fax: 410-955-5759.

Abstract

We used random mutagenesis and phenotypic rescue of adenylyl cyclase-null Dictyostelium cells to isolate loss-of-function mutations in the enzyme. Mutants were (i) catalytically inactive or (ii) resistant to chemoattractant receptor and guanosine 5′-3-O-(thio)triphosphate stimulation. Both classes of mutants harbored substitutions within the cytoplasmic C1a domain. Mutations that inactivated the enzyme were often at highly conserved positions. Those that blocked activation were grouped in two distinct regions: one close to the plane of the plasma membrane and another halfway within the C1 loop. Missense mutations or deletions within the transmembrane domains resulted in missorting of the protein. Our screen provides a simple and efficient method to separately define the sites of catalysis and regulation of this important class of enzymes.

Footnotes

↵^§ Recipient of a fellowship from the Medical Research Council of Canada.
↵¹ A. G. Gilman, personal communication.
↵* This work was supported by American Cancer Society Grant DB-1c. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵² The abbreviations used are:

ACA

adenylyl cyclase expressed during aggregation

GTPS

guanosine 5′-3-O-(thio)triphosphate

PCR

polymerase chain reaction

WT

wild type.
↵³Z. Xiao and P. N. Devreotes, manuscript in preparation.
- Received July 21, 1995.
- Revision received August 11, 1995.