The Carboxy-Terminal Domain of Gsot Is Necessary for Anchorage of the Activated Form in the Plasma Membrane

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the ot/3"y-type. In the activated state, the o~ subunit dissociates from the/3-y complex but remains anchored in the membrane. The c~ 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 ot subunit of Gs (Gso0, the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gset, we used a reconstitution model in which posttranslational addition of in vitro-translated Gsot to cycmembranes (obtained from a mutant of $49 cell line which does not express Gsu) restores the coupling between the/3-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 ot 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 Gso¢ whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated ot subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gst~ are required for association of this protein with the membrane. S IGNAL-TRANSDUCING GTP-binding proteins represent a family of heterotrimeric proteins (ot/33~-type) which transduce an extracellular signal recognized by a membrane receptor to an intracellular effector. Stimulation of the receptor induces the replacement of GDP by GTP on the c~ subunit, promoting the dissociation of the GTP-liganded c~ subunit from the/33, subunits and the subsequent activation of the effector by the c~ subunit (2, 4, 8, 21). The specificity of the interaction with the receptor and the effector is assigned to the ct subunit which contains the nucleotide binding site. Consequently, the c~ subunits of various GTP-binding proteins share homologous domains related to the common features of the transduction process such as the nucleotide binding site and eventually the site for interaction with the/3~, subunits. Alternatively, divergent domains are expected to specify the unique properties of each a subunit such as the receptor-binding domain or the effector-activating domain (2, 4, 8, 21). Divergence may also affect some properties such as membrane anchorage of the activated ~ subunit when it is dissociated from the /3")' complex. For example, visual phototransduction is carried out by a GTP-binding protein, transducin (T), l which is not strongly associated with the 1. Abbreviations used in this paper: Gi, the guanine nucleotide-binding protein which mediates inhibition of adenylate cyclase; Go, a new GTP-binding protein from membranes of bovine brain; Gs, the guanine nucleotidemembrane (15); moreover the GTP form of Tct subunit can be released from the disk membrane at 100 mM salt. Although it is very homologous to the Tc~ subunit, the subunit of Go (22, 26) and the three Gi subtypes (3, 11) was shown to contain a myristic acid on the amino-terminal glycine (5). Trypsin digestion of GTP'rS-treated membranes, from brain and neutrophil, removed a 2-kD amino-terminal domain on the ot subunit of Go or Gi and the proteolytic deletion was accompanied by the release of the tryptic large fragment from the membrane (6). More recently, amino-terminal myristoylation of these proteins was shown to be essential for their membrane attachment (12, 19), suggesting that the amino-terminal myristic acid may be sufficient for the anchorage in the membrane of the activated form of these GTPbinding proteins. Therefore, the domains involved in the interaction with the membrane, as well as the nature and the location of posttranslational modifications which contribute to or assign the membrane association, may differ from one GTP-binding protein to another. Although the a subunit of Gs is not myristoylated (5), the interaction of Gsa with the membrane of wild-type $49 cells binding protein which mediates activation of adenylate cyclase; GTPvS, guanosine 5'-(3-O-thio)-triphosphate; T, transducin from bovine rod outer segments. © The Rockefeller University Press, 0021-9525/90/10/1427/9 $2.00 The Journal of Cell Biology, Volume 111, October 199