ANALYSIS OF THE N-TERMINAL BINDING DOMAIN OF G(0)ALPHA

Signalling from membrane receptors through heterotrimeric G-proteins ( G alpha and G beta gamma) to intracellular effecters is a highly regul ated process. Receptor activation causes exchange of GTP for GDP on G alpha and dissociation of G alpha from G beta gamma. Both subunits rem ain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of G alpha subunits interacts with the membrane by several partially defin ed mechanisms: the anchoring of G alpha to the more hydrophobic G beta gamma subunits, the interaction of N-terminal lipids (palmitate and/o r myristate) with the membrane, and attachment of amino acid regions t o the membrane {amino acids 11-14 of G(o)a (D[11-14]); Busconi, Boutin and Denker (1997) Biochem, J. 323, 239-244}. We characterized N-termi nal mutants of G(o) alpha with known G beta gamma-binding properties f or the ability to interact with phospholipid vesicles and membranes pr epared from cultured cells (acceptor membranes). In vitro analysis all ows membrane interactions that are important to the activated and depa lmitoylated state of G alpha to be characterized. Subcellular localiza tion was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylati on do not account for changes in membrane binding. Disrupting the N-te rminal alpha-helix of G(o) alpha with a proline point mutation at Arg- 9 (R9P) does not affect interactions with G beta gamma on sucrose-dens ity gradients but significantly reduces acceptor membrane binding. Del etion of amino acids 6-15 (D[6-15]; reduced G beta gamma binding) or d eletion of amino acids 3-21 (D[3-21]); no detectable G beta gamma bind ing) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-t ype G(o) alpha as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoy lation is detected. The binding of G(o) alpha and mutants translated i n vitro to liposomes indicates that Go alpha preferentially binds to n eutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to p hosphatidylcholine liposomes like G(o) alpha, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interac tions of the N-terminus of G alpha subunits with the membrane may be a ffected by both membrane proteins and lipids. A detailed understanding of G alpha-membrane interactions may reveal unique mechanisms for reg ulating signal transduction.