The second cytoplasmic loop of metabotropic glutamate receptor functions at the third loop position of rhodopsin.

G protein-coupled receptors identified so far are classified into at least three major families based on their amino acid sequences. For the family of receptors homologous to rhodopsin (family 1), the G protein activation mechanism has been investigated in detail, but much less for the receptors of other families. To functionally compare the G protein activation mechanism between rhodopsin and metabotropic glutamate receptor (mGluR), which belong to distinct families, we prepared a set of bovine rhodopsin mutants whose second or third cytoplasmic loop was replaced with either the second or third loop of Gi/Go- or Gq-coupled mGluR (mGluR6 or mGluR1). Among these mutants, the mutants in which the second or third loop was replaced with the corresponding loop of mGluR exhibited no G protein activation ability. In contrast, the mutant whose third loop was replaced with the second loop of Gi/Go-coupled mGluR6 efficiently activated Gi but not Gt: this activation profile is almost identical with those of the mutant rhodopsins whose third loop was replaced with those of the Gi/Go-coupled receptors in family 1 [Yamashita et al. (2000) J. Biol. Chem. 275, 34272-34279]. The mutant whose third loop was replaced with the second loop of Gq-coupled mGluR1 partially retained the Gi coupling ability of rhodopsin, which is in contrast to the fact that all the rhodopsin mutants having the third loops of Gq-coupled receptors in family 1 exhibit no detectable Gi activation. These results strongly suggest that the molecular architectures of rhodopsin and mGluR are different, although the G protein activation mechanism involving the cytoplasmic loops is common.     

Functional interaction between bovine rhodopsin and G protein transducin

To elucidate the mechanisms of specific coupling of bovine rhodopsin with the G protein transducin (G(t)), we have constructed the bovine rhodopsin mutants whose second or third cytoplasmic loop (loop 2 or 3) was replaced with the corresponding loop of the G(o)-coupled scallop rhodopsin and investigated the difference in the activation abilities for G(t), G(o), and G(i) among these mutants and wild type. We have also prepared the Galpha(i) mutants whose C-terminal 11 or 5 amino acids were replaced with those of Galpha(t), Galpha(o), and Galpha(q) to evaluate the role of the C-terminal tail of the alpha-subunit on the specific coupling of bovine rhodopsin with G(t). Replacement of loop 2 of bovine rhodopsin with that of the scallop rhodopsin caused about a 40% loss of G(t) and G(o) activation, whereas that of loop 3 enhanced the G(o) activation four times with a 60% decrease in the G(t) activation. These results indicated that loop 3 of bovine rhodopsin is one of the regions responsible for the specific coupling with G(t). Loop 3 of bovine rhodopsin discriminates the difference of the 6-amino acid sequence (region A) at a position adjacent to the C-terminal 5 amino acids of the G protein, resulting in the different activation efficiency between G(t) and G(o). In addition, the binding of region A to loop 3 of bovine rhodopsin is essential for activation of G(t) but not G(i), even though the sequence of the region A is almost identical between Galpha(t) and Galpha(i). These results suggest that the binding of loop 3 of bovine rhodopsin to region A in Galpha(t) is one of the mechanisms of specific G(t) activation by bovine rhodopsin.     

The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G(t)). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G(t) are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the beta(2)-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys(322) and Cys(323). We suggest that the amino terminus of the fourth loop interacts directly with G(t), particularly with Galpha(t), and with other regions of the intracellular surface of rhodopsin to support G(t) binding.     

Chimeric microbial rhodopsins containing the third cytoplasmic loop of bovine rhodopsin

G-protein-coupled receptors transmit stimuli (light, taste, hormone, neurotransmitter, etc.) to the intracellular signaling systems, and rhodopsin (Rh) is the most-studied G-protein-coupled receptor. Rh possesses an 11-cis retinal as the chromophore, and 11-cis to all-trans photoisomerization leads to the protein structural changes in the cytoplasmic loops to activate G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. Microbial rhodopsins possess an all-trans retinal, and all-trans to 13-cis photoisomerization triggers protein structural changes for each function. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. However, it was reported that bacteriorhodopsin (BR) chimeras containing the third cytoplasmic loop of bovine Rh are able to activate G-protein, suggesting a common mechanism of protein structural changes. Here we design chimeric proteins for Natronomonas pharaonis sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin), which has a two-orders-of-magnitude slower photocycle than BR. Light-dependent transducin activation was observed for most of the nine SRII chimeras containing the third cytoplasmic loop of bovine Rh (from Y223, G224, Q225 to T251, R252, and M253), but the activation level was 30,000–140,000 times lower than that of bovine Rh. The BR chimera, BR/Rh223-253, activates a G-protein transducin, whereas the activation level was 37,000 times lower than that of bovine Rh. We interpret the low activation by the chimeric proteins as reasonable, because bovine Rh must have been optimized for activating a G-protein transducin during its evolution. On the other hand, similar activation level of the SRII and BR chimeras suggests that the lifetime of the M intermediates is not the simple determinant of activation, because SRII chimeras have two-orders-of-magnitude's slower photocycle than the BR chimera. Activation mechanism of visual and microbial rhodopsins is discussed on the basis of these results.     

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.     

Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin.

Structural requirements for the activation of transducin by rhodopsin have been studied by site-specific mutagenesis of bovine rhodopsin. A variety of single amino acid replacements and amino acid insertions and deletions of varying sizes were carried out in the two cytoplasmic loops CD (amino acids 134-151) and EF (amino acids 231-252). Except for deletion mutant delta 137-150, all the mutants bound 11-cis-retinal and displayed normal spectral characteristics. Deletion mutant delta 236-239 in loop EF caused a 50% reduction of transducin activation, whereas deletion mutant delta 244-249 and the larger deletions in loop EF abolished transducin activation. An 8-amino acid deletion in the cytoplasmic loop CD as well as a replacement of 13 amino acids with an unrelated sequence showed no transducin activation. Several single amino acid substitutions also caused significant reduction in transducin activation. The conserved charged pair Glu-134/Arg-135 in the cytoplasmic loop CD was required for transducin activation; its reversal or neutralization abolished transducin activation. Three amino acid replacements in loop EF (S240A, T243V, and K248L) resulted in significant reduction in transducin activation. We conclude that 1) both the cytoplasmic loops CD and EF are required for transducin activation, and 2) effective functional interaction between rhodopsin and transducin involves relatively large peptide sequences in the cytoplasmic loops.     

Mechanism of rhodopsin kinase activation

The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.     

Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy.

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.     

Identification of putative sites of interaction between the human formyl peptide receptor and G protein.

Wild-type and 35 mutant formyl peptide receptors (FPRs) were stably expressed in Chinese hamster ovary cells. All cell surface-expressed mutant receptors bound N-formyl peptide with similar affinities as wild-type FPR, suggesting that the mutations did not affect the ligand-binding site. G protein coupling was examined by quantitative analysis of N-formyl-methionyl-leucyl-phenylalanine-induced increase in binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to membranes. The most prominent uncoupled FPR mutants were located in the N-terminal part of the second transmembrane domain (S63W and D71A) and the C-terminal interface of the third transmembrane domain (R123A and C124S/C126S). In addition, less pronounced uncoupling was detected with deletion mutations in the third cytoplasmic loop and in the cytoplasmic tail. Further analysis of some of the mutants that were judged to be uncoupled based on the [(35)S]GTPgammaS membrane-binding assay were found to transduce a signal, as evidenced by intracellular calcium mobilization and activation of p42/44 MAPK. Thus, these single point mutations in FPR did not completely abolish the interaction with G protein, emphasizing that the coupling site is coordinated by several different regions of the receptor. Mutations located in the putative fifth and sixth transmembrane domains near the N- and C-terminal parts of the third cytoplasmic loop did not result in uncoupling. These regions have previously been shown to be critical for G protein coupling to many other G protein-coupled receptors. Thus, FPR appears to have a G protein-interacting site distinct from the adrenergic receptors, the muscarinic receptors, and the angiotensin receptors.     

Rhodopsin determinants for transducin activation: a gain-of-function approach

Three cytoplasmic loops in the G protein-coupled receptor rhodopsin, C2, C3, and C4, have been implicated as key sites for binding and activation of the visual G protein transducin. Non-helical portions of the C2- and C3-loops and the cytoplasmic helix-8 from the C4 loop were targeted for a "gain-of-function" mutagenesis to identify rhodopsin residues critical for transducin activation. Mutant opsins with residues 140-148 (C2-loop), 229-244 (C3-loop), or 310-320 (C4-loop) substituted by poly-Ala sequences of equivalent lengths served as templates for mutagenesis. The template mutants with poly-Ala substitutions in the C2- and C3-loops formed the 500-nm absorbing pigments but failed to activate transducin. Reverse substitutions of the Ala residues by rhodopsin residues have been generated in each of the templates. Significant ( approximately 50%) restoration of the rhodopsin/transducin coupling was achieved with re-introduction of residues Cys140/Lys141 and Arg147/Phe148 into the C2 template. The reverse substitutions of the C3-loop residues Thr229/Val230 and Ser240/Thr242/Thr243/Gln244 produced a pigment with a full capacity for transducin activation. The C4 template mutant was unable to bind 11-cis-retinal, and the presence of Asn310/Lys311 was required for correct folding of the protein. Subsequent mutagenesis of the C4-loop revealed the role of Phe313 and Met317. On the background of Asn310/Lys311, the inclusion of Phe313 and Met317 produced a mutant pigment with the potency of transducin activation equal to that of the wild-type rhodopsin. Overall, our data support the role of the three cytoplasmic loops of rhodopsin and suggest that residues adjacent to the transmembrane helices are most important for transducin activation.     

Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues sub-divided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.     

Counterion displacement in the molecular evolution of the rhodopsin family

The counterion, a negatively charged amino acid residue that stabilizes a positive charge on the retinylidene chromophore, is essential for rhodopsin to receive visible light. The counterion in vertebrate rhodopsins, Glu113 in the third transmembrane helix, has an additional role as an intramolecular switch to activate G protein efficiently. Here we show on the basis of mutational analyses that Glu181 in the second extracellular loop acts as the counterion in invertebrate rhodopsins. Like invertebrate rhodopsins, UV-absorbing parapinopsin has a Glu181 counterion in its G protein-activating state. Its G protein activation efficiency is similar to that of the invertebrate rhodopsins, but significantly lower than that of bovine rhodopsin, with which it shares greater sequence identity. Thus an ancestral vertebrate rhodopsin probably acquired the Glu113 counterion, followed by structural optimization for efficient G protein activation during molecular evolution.     

The interaction with the cytoplasmic loops of rhodopsin plays a crucial role in arrestin activation and binding

The binding of arrestin to rhodopsin is initiated by the interaction of arrestin with the phosphorylated rhodopsin C-terminus and/or the cytoplasmic loops, followed by conformational changes that expose an additional high-affinity site on arrestin. Here we use an arrestin mutant (R175E) that binds similarly to phosphorylated and unphosphorylated, wild-type rhodopsin to identify rhodopsin elements other than C-terminus important for arrestin interaction. R175E-arrestin demonstrated greatly reduced binding to unphosphorylated cytoplasmic loop mutants L72A, N73A, P142A and M143A, suggesting that these residues are crucial for high-affinity binding. Interestingly, when these rhodopsin mutants are phosphorylated, R175E-arrestin binding is less severely affected. This effect of phosphorylation on R175E-arrestin binding highlights the co-operative nature of the multi-site interaction between arrestin and the cytoplasmic loops and C-terminus of rhodopsin. However, a combination of any two mutations disrupts the ability of phosphorylation to enhance binding of R175E-arrestin. N73A, P142A and M143A exhibited accelerated rates of dissociation from wild-type arrestin. Using sensitivity to calpain II as an assay, these cytoplasmic loop mutants also demonstrated reduced ability to induce conformational changes in arrestin that correlated with their reduced ability to bind arrestin. These results suggest that arrestin bound to rhodopsin is in a distinct conformation that is co-ordinately regulated by association with the cytoplasmic loops and the C-terminus of rhodopsin.     

NMR structure of the second intracellular loop of the alpha 2A adrenergic receptor: evidence for a novel cytoplasmic helix

A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR-G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on approximately 35-residue-long peptides including the critical second intracellular loop (i2) of the alpha 2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4-12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an alpha 2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the alpha 2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.     

Evidence for structural changes in carboxyl-terminal peptides of transducin alpha-subunit upon binding a soluble mimic of light-activated rhodopsin

Although a high-resolution crystal structure for the ground state of rhodopsin is now available, portions of the cytoplasmic surface are not well resolved, and the structural basis for the interaction of the cytoplasmic loops with the retinal G-protein transducin (G(t)) is still unknown. Previous efforts aimed at the design, construction, and functional characterization of soluble mimics for the light-activated state of rhodopsin have shown that grafting defined segments from the cytoplasmic region of bovine opsin onto a surface loop in a mutant form of thioredoxin (HPTRX) is sufficient to confer partial G(t) activating potential [Abdulaev et al. (2000) J. Biol. Chem. 275, 39354-39363]. To assess whether these designed mimics could provide a structural insight into the interaction between light-activated rhodopsin and G(t), the ability of an HPTRX fusion protein comprised of the second (CD) and third (EF) cytoplasmic loops (HPTRX/CDEF) to bind G(t) alpha-subunit (G(t)(alpha)) peptides was examined using nuclear magnetic resonance (NMR) spectroscopy. Transfer NOESY (TrNOESY) experiments show that an 11 amino acid peptide corresponding to the carboxyl terminus of G(t)(alpha) (GtP), as well as a "high-affinity" peptide analogue, HAP1, binds to HPTRX/CDEF in the fast-exchange regime and undergoes similar, subtle structural changes at the extreme carboxyl terminus. Observed TrNOEs suggest that both peptides when bound to HPTRX/CDEF adopt a reverse turn that is consistent with the C-cap structure that has been previously reported for the interaction of GtP with the light-activated signaling state, metarhodopsin II (MII). In contrast, TrNOESY spectra provide no evidence for structuring of the amino terminus of either GtP or HAP1 when bound to HPTRX/CDEF, nor do the spectra show any measurable changes in the CD and EF loop resonances of HPTRX/CDEF, which are conformationally dynamic and significantly exchange broadened. Taken together, the NMR observations indicate that HPTRX/CDEF, previously identified as a functional mimic of MII, is also an approximate structural mimic for this light-activated state of rhodopsin.     

Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin

BACKGROUND: The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-activated phosphorylated rhodopsin. RESULTS: We replaced eight residues of the EF loop of bacteriorhodopsin (BR) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were excised by V8 protease, revealing helices E and F in the AFM topographs. Rho EF was modeled onto the BR structure, and the envelope derived from the AFM data of IIIN was used to select probable models. CONCLUSIONS: A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.     

Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.     

Functionally discrete mimics of light-activated rhodopsin identified through expression of soluble cytoplasmic domains

Numerous studies on the seven-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal region in the binding and activation of proteins involved in visual transduction and desensitization. In our continuing studies on rhodopsin folding, assembly, and structure, we have attempted to reconstruct the interacting surface(s) for these proteins by inserting fragments corresponding to the cytoplasmic loops and/or the carboxyl-terminal tail of bovine opsin either singly, or in combination, onto a surface loop in thioredoxin. The purpose of the thioredoxin fusion is to provide a soluble scaffold for the cytoplasmic fragments thereby allowing them sufficient conformational freedom to fold to a structure that mimics the protein-binding sites on light-activated rhodopsin. All of the fusion proteins are expressed to relatively high levels in Escherichia coli and can be purified using a two- or three-step chromatography procedure. Biochemical studies show that some of the fusion proteins effectively mimic the activated conformation(s) of rhodopsin in stimulating G-protein or competing with the light-activated rhodopsin/G-protein interaction, in supporting phosphorylation of the carboxyl-terminal opsin fragment by rhodopsin kinase, and/or phosphopeptide-stimulated arrestin binding. These results suggest that specific segments of the cytoplasmic surface of rhodopsin can adopt functionally discrete conformations in the absence of the connecting transmembrane helices and retinal chromophore.