Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit

Abstract

Overall, these results demonstrated that R*-induced changes in G ␣ can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation. G-protein coupled receptors (GPCRs)3 represent a diverse group of seven transmembrane (TM) helix receptors that require agonist-dependent activation to initiate heterotrimeric (␣␤␥) G-protein-mediated intracellular signaling cascades. GPCR activation of cognate G-proteins are the first steps in cellular communication pathways responsible for signaling cascades that mediate vision, olfaction, taste, and the action of numerous hormones and neurotransmitters (1). Activation of a G-protein by its agonist-stimulated GPCR (R*) requires the propagation of structural signals from the receptor-binding interface to the guanine nucleotide-binding pocket. The structural basis for the interaction of a GPCR with its cognate G-protein and the subsequent activation of the G-protein by R* are not fully understood.Using signaling of the retinal G-protein transducin (G t ) by rhodopsin as a model system, we are applying solution NMR methods to track changes in the G-protein ␣-subunit (G ␣ ) associated with activated R* interactions. Rhodopsin, the rod cell photoreceptor involved in dimlight vision, represents one of the best studied GPCRs in terms of structure and function (2, 3). Photon capture triggers cis 3 trans isomerization of the retinal chromophore, which initiates structural changes in the TM helices resulting in the formation of the light-activated signaling state metarhodopsin II, R*. This is accompanied by functionally significant changes at the cytoplasmic surface that leads to the formation of binding and activation sites for several signaling proteins, including G t (4 -10). Crystal structures for the inactive (dark) state of rhodopsin have provided a detailed view of the retinal binding site and the cytoplasmic region (11-15). Although remarkably informative, the crystal structures provide few solid insights into the mechanism of signal transfer from R* to G t .Binding of heterotrimeric G-proteins to activated GPCRs requires the presence of both G ␣ and G-protein ␤␥-subunits (G ␤␥ ). The following three regions on the ␣-subunit of G t (G t␣ ) are known to be important for receptor interactions; the amino-terminal 23 residues, an internal sequence from amino acids 305-315, and the carboxyl-terminal 11 amino acids (16 -18). Upon binding to R*, G t␣ is thought to undergo structural changes in both the amino-and carboxyl-terminal regions. High resolution crystal structures of G ␣ subunits, including G t␣ (19 -24) 3 The abbreviations used are: GPCR, G-protein coupled receptor; TM, transmembrane; R*, the agonist activated form of a GPCR; GTP␥S, guanosine 5Ј-O-3-thiotriphosphate; CMC, critical micelle concentration; DM, n-dodecyl ␤-D-maltopyranoside; ChiT, prodomain released chimeric G ␣ ; ROS, rod outer segment; HSQC, heteronuclear single quantum correlation; HPTRX/CDEF, protein chimera containing segments from the CD and EF loops of bovine opsin g...

A Concerted Mechanism for Opening the GDP Binding Pocket and Release of the Nucleotide in Hetero-Trimeric G-Proteins

“…This rigid motion of the whole helical domain was also supported by other experimental data suggesting no strong structural internal rearrangement of this domain during activation. 38 Finally, the motion along mode 17 showed a kink of the α5 helix (Fig. 6b, on the right).…”

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

“…The R*⅐G␣CT structure likely shows part of the R*⅐G t [empty] complex (4), consistent with earlier EPR spectroscopic work (13). Comparison with the G t crystal structure (14) indicates a rotational and translational movement of the G t ␣ C-terminal ␣5 helix in R*⅐G t [empty], which is a structural perturbation in the G protein necessary for GDP release (4,13,(15)(16)(17). Kinetic analysis has suggested, and it will be further confirmed in this study, that GDP release is triggered by conversion of an intermediate complex (R*⅐G t ⅐GDP) into the nucleotide-free R*⅐G t [empty] complex (18).…”

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…The main points of open discussion have been delineated as follows: 1) How ligand activated GPCRs effect the guanine nucleotide exchange at Gα [36,37]; 2) Whether Gα, Gβγ, and GPCRs remain associated after activation [4,8,38,39]; 3) How the G protein subunits and their effectors are spatially arranged during signaling [6,40].…”