A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTPbinding proteins. How these receptors recognize and promote nucleotide exchange on G protein ␣ subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) ␣ subunit C-terminal undecapeptide Gt␣(340-350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gt␣(340-350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an ␣ L type. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325-346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor-G protein interface is demonstrated.

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Post-translational prenylation of the carboxyl-terminal cysteine is a characteristic feature of the guanine nucleotide-binding protein (G protein) ␥ subunits. Recent findings show that the farnesylated COOH-terminal tail of the ␥1 subunit is a specific determinant of rhodopsin-transducin coupling. We show here that when synthetic peptides specific to the COOH-terminal tail of ␥1 are chemically modified with geranyl, farnesyl, or geranylgeranyl groups and tested for their ability to interact with light activated rhodopsin, the farnesylated peptide is significantly more effective. These results show that an appropriate isoprenoid on the G protein ␥ subunit serves not only a membrane anchoring function but in combination with the COOH-terminal domain specifies receptor-G protein coupling. Heterotrimeric (␣␤␥) G proteins1 control key signaling pathways inside a cell by relaying information from activated transmembrane receptors to specific effector molecules (1-3). All known G protein ␥ subunits are modified by either a 15-carbon farnesyl or by a 20-carbon geranylgeranyl moiety (4, 5). The ␥1 subunit, which is associated with the G protein Gt, is farnesylated, whereas most other ␥ subunits are geranylgeranylated. Prenylation occurs on the cysteine in the COOH-terminal CAAX motif (C is a cysteine, A is an aliphatic amino acid, X is any amino acid), and the nature of the last residue is thought to determine farnesylation or geranylgeranylation.Prenylation of the ␥ subunits has been shown to be necessary for G protein membrane attachment (6 -8). There is also evidence that suggests the involvement of the prenyl group in protein-protein interactions (9 -14). However, it is unclear why G protein ␥ subunits are modified by two different types of isoprenoids. Studies of some prenylated proteins (rhodopsin kinase (10), p21 (11), yeast a-factor (13)) demonstrate that the biological consequences of differential prenylation may be more complex than anticipated. It has also been difficult to examine the role of the different isoprenoids because recombinant proteins with altered CAAX domains seem to be prenylated with a mixture of farnesyl and geranylgeranyl (13,15). To circumvent this problem, we have used peptides chemically modified with various isoprenoids, since we could confirm appropriate prenylation accurately in each case by mass spectrometry.To identify the role of the isoprenoid, we have examined peptides chemically modified with farnesyl, geranyl (C-10), and geranylgeranyl, for their relative efficacies at stabilizing light activated rhodopsin. We have recently shown that the farnesylated COOH-terminal domain of the ␥1 subunit directly interacts with an activated receptor (light-activated rhodopsin) and is important for the holomeric G protein to effectively couple with rhodopsin (12, 16). Both the farnesyl group and the appropriate primary structure of the COOH-terminal domain of ␥1 are essential for effective rhodopsin-Gt interaction. By assaying farnesylated peptides of varying lengths we have identified here the ...

Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein-protein interactions catalyzing the activation of the G-protein transducin (Gt). Using stabilization and photoregeneration of the receptor's signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt ␥-subunit, Gt␥(50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gt␤␥ complex in direct activation assays. Competition by Gt␥(50-71)farnesyl with Gt for the active receptor suggests a pivotal role for Gt␤␥ in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt ␣-subunit, Gt␣(340-350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein.In the center of the model is specific recognition of conformationally distinct sites of R* by Gt␣(340-350) and Gt␥(50-71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and͞or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.Rhodopsin is a prototypical G-protein-coupled receptor in retinal rods (1, 2). Available information supports a mechanism in which the initial isomerization of the chromophore 11-cis-retinal, and thus the formation of the agonistic all-transretinal, leads to crucial contacts between the ligand and the apoprotein opsin. These steric constraints result in a defined arrangement of donor and acceptor groups for proton translocations leading to subsequent tautomeric conformations of the receptor, identified as ''metarhodopsin'' photointermediates, each with a characteristic absorption spectrum. Metarhodopsin I (MI, max ϭ 478 nm) is in a pH-and temperaturedependent equilibrium with metarhodopsin II (MII, max ϭ 380 nm), distinguished by its deprotonated Schiff base linkage [and broken salt bridge (3, 4)] between the retinal and Lys 296 . MII has been shown to catalyze retina rod cell-specific Gprotein (Gt) activation through nucleotide exchange (5, 6).Despite recent progress in structure determination of both Gt and rhodopsin, the molecular mechanism of signal transfer between the two proteins is poorly understood. Interacting surfaces of rhodopsin and Gt include intracellular loops of the receptor and domains on both Gt ␣-and Gt ␥-subunits (1, 7-9). C-terminal domains of Gt ␣-and Gt ␥-subunits, Gt␣(340-350) and...