Activating mutations in the NH2- and COOH-terminal moieties of the Gsα subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation
✍ Scribed by Sunil K. Gupta; N. Dhanasekaran; Lynn E. Heasley; Gary L. Johnson
- Book ID
- 102878629
- Publisher
- John Wiley and Sons
- Year
- 1991
- Tongue
- English
- Weight
- 834 KB
- Volume
- 47
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
The a subunit polypeptides of the G proteins G, and G,, stimulate and inhibit adenylyl cyclase, respectively. The a, and a,, subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH-terminal moiety of the a, polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH,-terminal half of the a, polypeptide encodes domains regulating py interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH,-or COOH-terminal coding sequence of a, substituted with the corresponding sequence were used to introduce multi-residue non-conserved mutations in different domains of the a, polypeptide. Mutation of either the amino-or carboxy-terminus results in an a, polypeptide which constitutively activates CAMP synthesis when expressed in Chinese hamster ovary cells. The activated a, polypeptides having mutations in either the NH,or COOH-terminus demonstrate an enhanced rate of GTPyS activation of adenylyl cyclase. In membrane preparations from cells expressing the various a, mutants, COOH-terminal mutants, but not NH,-terminal a, mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTPyS and fluoride ion. Neither mutation at the NH,-nor COOH-terminus had an effect on the GTPase activity of the a, polypeptides. Thus, mutation at NH,-and COOH-termini influence the rate of a, activation, but only the COOH-terminus appears to be involved in the regulation of the a, polypeptide activation domain that interacts with adenylyl cyclase.