Heterotrimeric guanine nucleotide binding proteins (G-proteins) transmit signals from membrane receptors to a variety of intracellular effectors. G-proteins reversibly associate with components of the signal transduction system, yet remain membrane attached throughout the cycle of activation. The Gα subunits remain attached to the plasma membrane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of Gα subunits are involved in membrane binding. We used in vitro translation, a technique widely utilized to characterize functional aspects of G-proteins, and interactions with donor-acceptor membranes to demonstrate that amino acids 11-14 of Gαo contribute to membrane binding. The membrane binding of Gαo lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Gαo. Several other N-terminal mutants of Gαo were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and βγ interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Gαo and mutants was characterized in transiently transfected 35S-labelled and [3H]myristate-labelled COS cells, amino acids 11-14 contributed to membrane binding. These studies reveal that membrane binding of Gα subunits occurs by a combination of factors that include lipids and amino acid sequences. These regions may provide novel sites for interaction with membrane components and allow additional modulation of signal transduction.
Present address: Instituto Investiqaciones Biologicas, Universidad Nacional Mar del Plata, Casilla de Correo 1245, 7600-Mar del Plata, Argentina.