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...

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The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G t ), is not well defined. We developed a novel assay to measure the ability of G t , or G t -derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340 -350 of the ␣ subunit of G t , or a cysteinylthioetherfarnesyl peptide corresponding to residues 50 -71 of the ␥ subunit of G t , are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the ␥-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G t (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930Chem. 275, -1936. Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the ␣ subunit of G t . We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the ␣ subunit of G t and plays a role in the regulation of ␤␥ subunit binding.G protein-coupled receptors transmit extracellular signals to the cell's interior via heterotrimeric G proteins and effector enzymes or ion channels (1, 2). Rhodopsin is one of the archetypes of the G protein-coupled receptor superfamily. It triggers the biochemical amplification machinery of the visual cascade in the rod photoreceptor cell, which comprises the G protein transducin (G t ) 1 and the effector, a cyclic GMP-specific phosphodiesterase (3, 4). The transduction of a light signal begins with the photochemical cis-trans isomerization of the chromophore, 11-cis-retinal. Protein conformational changes are transmitted from the ligand-binding site to the cytoplasmic surface of the receptor where catalytic activation of G t occurs. This intramolecular conversion from inactive (rhodopsin) to active (R*) states mediated by chromophore isomerization has been termed "signal transmission" (5). Key structural correlates of the transition to the active state include deprotonation of the retinylidene Schiff base (6) with concomitant protonation of the Glu 113 counterion (7,8) and the protonation of the cytoplasmic surface of rhodopsin (9, 10) mediated by the highly conserved Glu 134 residue at the cytoplasmic border of transmembrane (TM) helix 3. Movements of TM helices have been proposed to accompany the signal transmission process, with a change in the orientation of TM helices 3 and 6 relative to each other as the most prominent feature (11-13).The cytoplasmic surface of rhodopsin comprises four loops and a carboxyl-terminal tail. The first (C1), second (C2), and third (C3) cytoplasmic loops connect adjacent TM helices. The fourth cytoplasmic loop (C4) is bounded by TM helix 7 at its ...

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After lightinduced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.The retinal photoreceptor rhodopsin is one of the archetypes of the G-protein-coupled receptor superfamily. It is composed of the apoprotein opsin comprising seven transmembrane helices (TMs) 1 and the chromophore 11-cis-retinal, which is covalently bound to Lys 296 in TM7 via a protonated Schiff base (SB), keeps the receptor in the inactive conformation, and acts as a highly effective inverse agonist. Following the absorption of a photon, the retinal isomerizes to a strained all-trans-conformation (1), which induces a series of conformational rearrangements of the opsin moiety. The final product of this reaction sequence is the active conformation (R*) that contains all-trans-retinal as a covalently bound agonist and is capable of catalyzing the activation of the retinal G-protein transducin (G t ) (for reviews see Refs. 2-4).These events can be summarized as shown in Fig. 1A). After cis-trans-isomerization and initial, short lived intermediates, illuminated rhodopsin progresses from the lumirhodopsin form to the first long lived state, metarhodopsin I (meta I). Up to and including meta I, the SB of the pigment remains protonated. Deprotonation of the SB and protonation of its counterion Glu 113 mark the transition from the meta I ( max ϭ 478 nm) to the metarhodopsin II (meta II) conformation (5), which is characterized by a strongly blue-shifted absorbance maximum ( max ϭ 380 nm). SB deprotonation is known to be necessary for interaction with G t (6 -8) and is accompanied by the uptake of a proton from the aqueous solution (9 -11). pH rate profiles of G t activation and proton uptake measurements have shown the opsin residue Glu 134 to be an important mediator of the uptake or even the acceptor of this proton (12, 13). Based on computer ...

The method of differential equations has been proven to be a powerful tool for the computation of multi-loop Feynman integrals appearing in quantum field theory. It has been observed that in many instances a canonical basis can be chosen, which drastically simplifies the solution of the differential equation. In this paper, an algorithm is presented that computes the transformation to a canonical basis, starting from some basis that is, for instance, obtained by the usual integration-by-parts reduction techniques. The algorithm requires the existence of a rational transformation to a canonical basis, but is otherwise completely agnostic about the differential equation. In particular, it is applicable to problems involving multiple scales and allows for a rational dependence on the dimensional regulator. It is demonstrated that the algorithm is suitable for current multi-loop calculations by presenting its successful application to a number of non-trivial examples.

The integration of differential equations of Feynman integrals can be greatly facilitated by using a canonical basis. This paper presents the Mathematica package CANONICA, which implements a recently developed algorithm to automatize the transformation to a canonical basis. This represents the first publicly available implementation suitable for differential equations depending on multiple scales. In addition to the presentation of the package, this paper extends the description of some aspects of the algorithm, including a proof of the uniqueness of canonical forms up to constant transformations.

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