N-terminal binding domain of Galpha subunits: involvement of amino acids 11-14 of Galphao in membrane attachment.

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 Galpha 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 Galpha 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 Galphao contribute to membrane binding. The membrane binding of Galphao lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Galphao. Several other N-terminal mutants of Galphao were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and betagamma interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Galphao 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 Galpha 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.