The carboxy-terminal domain of Gsα is necessary for anchorage of the activated form in the plasma membrane

Abstract

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the αβγ-type. In the activated state, the α subunit dissociates from the βγ complex but remains anchored in the membrane. The α subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the α subunit of Gs (Gsα), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gsα, we used a reconstitution model in which posttranslational addition of in vitro-translated Gsα to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gsα) restores the coupling between the β-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated α chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gsα whereas proteolytic removal of a 1-kD carboxy-terminal domain abolished membrane interaction. Thus, in contrast to the myristoylated α subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gsα are required for association of this protein with the membrane.