How a G Protein Binds a Membrane

Abstract

Heterotrimeric G proteins interact with receptors and effectors at the membrane-cytoplasm interface. Structures of soluble forms have not revealed how they interact with membranes. We have used electron crystallography to determine the structure in ice of a helical array of the photoreceptor G protein, transducin, bound to the surface of a tubular lipid bilayer. The protein binds to the membrane with a very small area of contact, restricted to two points, between the surface of the protein and the surface of the lipids. Fitting the x-ray structure into the membrane-bound structure reveals one membrane contact near the lipidated G␥ C terminus and G␣ N terminus, and another near the G␣ C terminus. The narrowness of the tethers to the lipid bilayer provides flexibility for the protein to adopt multiple orientations on the membrane, and leaves most of the G protein surface area available for protein-protein interactions.G proteins mediate signal transduction pathways on the membranes of virtually all eukaryotic cells. In the inactive, GDP-bound, heterotrimeric state most G proteins are themselves tightly membrane associated. Activation takes place when GTP is exchanged for GDP in a complex with an integral membrane G protein-coupled receptor, which produces separated and active G␣-GTP and G␤␥ proteins. These active subunits are then free to move along the membrane and regulate their downstream effectors that may be either peripherally membrane associated (e.g. cGMP phosphodiesterase, phospholipase C) or integral membrane proteins (e.g. adenylyl cyclase and ion channels).There have been numerous studies of G protein-membrane interactions using biochemical, functional, and mutagenic approaches. These have made it clear that membrane association and localization play a critical role in G protein function and have also revealed biochemical features of G␣ and G␥ that are important for membrane binding. All G␣ subunits have one or more fatty acids added at or near their N termini, and both the fatty acids and the amino acids in the N-terminal region are important for membrane binding (1-3). The attachment site is either an N-terminal glycine, which is linked to fatty acid, usually myristic acid, in an amide linkage, or a cysteine at position 2, which is linked to palmitic acid in a thioester linkage. The G␥ subunits are proteolytically processed to leave a C-terminal methyl-esterified cysteine residue linked via a thioether to either a 15-or 20-carbon isoprenyl chain (4 -8).Forms of G␥ without these modifications have been studied, and they show reduced interactions with membranes and defective signaling properties (9 -14).In the crystal structures of G protein heterotrimers (15, 16) these hydrophobic modifications and their immediately adjacent amino acids are missing; however, the resolved portions of the G␥ C terminus and the G␣ N terminus are close to one another in space, suggesting a common site for membrane insertion. The only G protein structure in which a lipid modification is present and visible is the complex o...

The Importance of N-terminal Polycysteine and Polybasic Sequences for G14α and G16α Palmitoylation, Plasma Membrane Localization, and Signaling Function

“…Metabolic Labeling and Immunoprecipitation-Cells were transfected at a density of 7.125 ϫ 10 6 cells/plate in 10-cm 2 plates with a total of 24 g of DNA/plate. For experiments with the NTG␣-EGFP-FLAG fusion proteins, 24 g of the fusion protein of interest was transfected per plate.…”

“…For experiments with the NTG␣-EGFP-FLAG fusion proteins, 24 g of the fusion protein of interest was transfected per plate. For experiments with full-length G␣, cells were transfected with 16.8 g of EEtagged G␣ Ϯ C/S, 4.8 g of G␤ 1 , and 2.4 g of G␥ 2 . Twenty four hours after transfection, cells were labeled and immunoprecipitated according to a method described in detail elsewhere with minor modifications (27,28).…”

“…Both the G␣ and G␥ subunits of the heterotrimer bind the lipid bilayer to position the G protein for activation by cognate G protein-coupled receptors (1,2). In response to receptor-induced activation with GTP, the G protein subunits redistribute and selectively stimulate membrane-bound effector molecules (reviewed in Refs.…”

“…3,4). The four G␣ subunits of the G q family, G q ␣, G 11 ␣, G 14 ␣, and G 16 ␣, transfer receptor-generated signals to phospholipase C-␤ isozymes 1-4 (PLC-␤1-4) 2 (reviewed in Ref. 5) and other protein binding partners (reviewed in Ref.…”

Regulatory Interactions between the Amino Terminus of G-protein βγ Subunits and the Catalytic Domain of Phospholipase Cβ2

“…Second, the N20K region within the Y-domain of PLC␤ is within close proximity to the substrate binding site of the enzyme (14). Recent low resolution structures of G␤␥ bound to membrane tubules place the N-terminal region of G␤␥ far from the membrane surface (30). Therefore, if the Y-domain of PLC␤ is bound to the N terminus, it would be sequestered from its substrate.…”