Functional Domains of the Gs α Subunit: Role of the C-Terminus in the Receptor-Dependent and Receptor-Independent Activation

Abstract

We have developed a rapid and simple model for studying functional domains of Gs α subunit, the GTP binding protein involved in adenylyl cyclase activation. Cyc⁻ membranes prepared from a variant S49 cell line which does not express the α subunit of Gs are reconstituted by the in vitro translated Gs α subunit. Since the messenger RNA used for in vitro translation is generated from in vitro transcription of the cDNA encoding Gs α subunit, it is possible to introduce genetic modifications at the nucleotide level and analyze their consequences at the amino-acid level on the functional properties of the protein. We have constructed mutated α chains which correspond to various deletions of the carboxy-terminal region. Removal of the 9 carboxy-terminal residues uncoupled the α subunit from the receptor whereas deletion of the 26 carboxy-terminal residues blocked any activation induced either in a receptor-dependent or in a receptor-independent manner.