N-TERMINAL BINDING DOMAIN OF G-ALPHA SUBUNITS - INVOLVEMENT OF AMINO-ACIDS-11-14 OF G-ALPHA(O) IN MEMBRANE ATTACHMENT

Heterotrimeric guanine nucleotide binding proteins (G-proteins) transm it signals from membrane receptors to a variety of intracellular effec ters. G-proteins reversibly associate with components of the signal tr ansduction system, yet remain membrane attached throughout the cycle o f activation. The G alpha subunits remain attached to the plasma membr ane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of G alpha subunits are involved in membrane binding. We used in vitro tran slation, a technique widely utilized to characterize functional aspect s of G-proteins, and interactions with donor-acceptor membranes to dem onstrate that amino acids 11-14 of G alpha(o) contribute to membrane b inding. The membrane binding of G alpha(o) lacking amino acids 11-14(D [11-14]) was significantly reduced at all membrane concentrations in c omparison with wild-type G alpha(o). Several other N-terminal mutants of G alpha(o) were characterized as controls, and these results indica te that differences in myristoylation, palmitoylation and beta gamma i nteractions do not account for the reduced membrane binding of D[11-14 ]. Furthermore, when membrane attachment of G alpha(o) and mutants was characterized in transiently transfected S-35-labelled and [H-3]myris tate-labelled COS cells, amino acids 11-14 contributed to membrane bin ding. These studies reveal that membrane binding of G alpha subunits o ccurs by a combination of factors that include lipids and amino acid s equences. These regions may provide novel sites for interaction with m embrane components and allow additional modulation of signal transduct ion.