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...