Tertiary Interactions between the Fifth and Sixth Transmembrane Segments of Rhodopsin

To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M 3 muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M 3 muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M 3 muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532 7.42 ) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142 3.27 -Asn-152 3.37 . Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151 3.36 C and Cys-532. A three-dimensional model of the inactive state of the M 3 muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.

Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of an Agonist-induced Conformational Change Occurring Adjacent to the Ligand-binding Pocket of the M3 Muscarinic Acetylcholine Receptor

Han

Hamdan

Kim

et al. 2005

“…These facts, coupled with our new results, enable us to speculate that the amino acid pair at positions 269 and 208 would work as a pivot to induce the outward shift in the cytoplasmic region of helix VI (Fig. 6), whereas its extracellular region does not move significantly (18,19).…”

Section: Identification Of the Amino Acid Residue(s) Whose Mutation Csupporting

confidence: 63%

A Pivot between Helices V and VI near the Retinal-binding Site Is Necessary for Activation in Rhodopsins

Tsukamoto¹,

Terakita²,

Shichida

2010

Rhodopsins are photoreceptor proteins that have diverged from ligand-binding G protein-coupled receptors (GPCRs). Unlike other GPCRs, rhodopsins contain an intrinsic antagonist, 11-cis-retinal, which is converted to the agonist all-transretinal upon absorption of a photon. Through evolution, vertebrate rhodopsins have lost the ability of direct binding to the agonist, but some invertebrate and vertebrate non-visual rhodopsins have retained this ability. Here, we investigated the difference in the agonist-binding state between these rhodopsins to further our understanding of the structural and functional diversity of rhodopsins. Mutational analyses of agonist-binding rhodopsin showed that replacement of Ala-269, one of the residues constituting the antagonist-binding site, with bulky amino acids resulted in a large spectral shift in its active state and a great reduction in G protein activity, whereas these were rescued by subsequent replacement of Phe-208 with smaller amino acids. Although similar replacements in vertebrate rhodopsin did not cause a spectral shift in the active state, a similar reduction in and recovery of G protein activity was observed. Therefore, the agonist is located close to Ala-269 in the agonist-binding rhodopsin, but not in vertebrate rhodopsins, and Ala-269 with Phe-208 acts as a pivot for the formation of the G proteinactivating state in both rhodopsins. The positions corresponding to Ala-269 and Phe-208 in other GPCRs have been reported to form part of an agonist-binding site. Therefore, an agonistbinding rhodopsin has the molecular architecture of the agonist-binding site similar to that of a general GPCR, whereas vertebrate rhodopsins changed the architecture, resulting in loss of agonist binding during molecular evolution.Rhodopsin is a member of the family of G protein-coupled receptors (GPCRs) 4 that have a seven-transmembrane ␣-helical structure. It contains the antagonist (inverse agonist) 11-cisretinal covalently attached to its protein moiety, opsin. The agonist of rhodopsin is all-trans-retinal, but curiously, vertebrate rhodopsins have no ability to bind this agonist directly (1). Instead, the agonist all-trans-retinal is produced by photoisomerization of the antagonist 11-cis-retinal in the rhodopsin molecule upon photon absorption. Loss of the ability to directly bind exogenous agonist is probably the result of molecular evolution of vertebrate rhodopsin, which would have favored the decrease in background "noise" in the dark state (2). We have found that, in contrast to vertebrate rhodopsins, a non-vertebrate rhodopsin, amphioxus rhodopsin (3), and a vertebrate pineal UV light-sensitive pigment, lamprey parapinopsin 5 (4), still have the ability to bind the agonist directly, and the active state formed by direct binding of agonist exhibited biochemical and spectroscopic properties indistinguishable from those of the photoproduct that was formed by irradiation of the antagonist-binding rhodopsin. In addition, the G protein-activating efficiencies of these rhodopsins we...

“…The resulting trajectories were concate-nated and used to produce the final refined model of the CLR. The active state of the CLR was achieved through the use of a modified rhodopsin template, which was consistent with experimentally derived distance restraints obtained from the literature (21)(22)(23)(24)(25). Homology models of the active CLR were refined through the use of MD simulations as described above.…”

Section: Methodsmentioning

confidence: 55%

The Second Intracellular Loop of the Calcitonin Gene-related Peptide Receptor Provides Molecular Determinants for Signal Transduction and Cell Surface Expression

Conner

Simms

Howitt

et al. 2006

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to G s , but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-proteincoupled receptors in general. G-Protein coupled receptors (GPCRs)2 comprise a large superfamily of membrane-spanning proteins encoded by 2-3% of the human genome. These receptors respond to an incredibly diverse array of stimuli from odorants, amines, peptides, and light through a series of broadly similar activation mechanisms and accessory proteins. The pharmaceutical implications of understanding these proteins cannot be underestimated, and the crystal structure of rhodopsin has presented many new avenues of study for this family (1).Several structural and functional motifs are well characterized within the intracellular domains of the largest known family (A) of GPCRs. These include the conserved (D/E)R(Y/H) motif on the boundary between transmembrane helix 3 and the second intracellular loop (ICL2) and which is well documented to have a significant role in the activation mechanism (2) and the NPXXY motif of TM7, which can have diverse roles in the constitutive phosphorylation, internalization, and signaling of many family A GPCRs (3, 4). In addition, residues within ICL2 juxtaposed to transmembrane helix 3 have been shown to be involved in receptor stability (5).In contrast, much less is known about the important amino acids of family B receptors, which include the calcitonin family of receptors and which tend to bind larger peptide...