Cytoplasmic Loops Of Rhodopsin Research Articles

A study of the photoinduced conformational mobility of spin-labeled regenerated rhodopsin by ESR spectroscopy

Saturation transfer EPR spectroscopy is used to study the photoinduced conformational changes of spin-labeled (Cys140 and Cys316) rhodopsin in a photoreceptor membrane. Illumination of rhodopsin with “yellow” light (λ > 450 nm), converting it from the dark form into metarhodopsin II, a physiologically active form, is accompanied by an increase in the mobility of the cytoplasmic loop (Cys140) and eighth alpha-helix (Cys316), whereas the subsequent illumination with “blue” light (λ < 445 nm), which converts metarhodopsin II into a mixture of inactive photoregenerated products, results in a decrease in the mobility. The restoration of the specific features of the photoinduced changes in the conformational mobility of the cytoplasmic loops of rhodopsin regenerated in the dark is demonstrated.

Recoverin Binds Exclusively to an Amphipathic Peptide at the N Terminus of Rhodopsin Kinase, Inhibiting Rhodopsin Phosphorylation without Affecting Catalytic Activity of the Kinase

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.

Light-Driven Activation of β2-Adrenergic Receptor Signaling by a Chimeric Rhodopsin Containing the β2-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.

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.

Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent.

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at lambda > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the T(alpha) subunit. Crosslinking to T(alpha) was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in T(alpha) were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19-Arg-28 at the N-terminal region of T(alpha). The total results show that both the N and the C termini of T(alpha) are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.

Distinct Roles of the Second and Third Cytoplasmic Loops of Bovine Rhodopsin in G Protein Activation

In contrast to the extensive studies of light-induced conformational changes in rhodopsin, the cytoplasmic architecture of rhodopsin related to the G protein activation and the selective recognition of G protein subtype is still unclear. Here, we prepared a set of bovine rhodopsin mutants whose cytoplasmic loops were replaced by those of other ligand-binding receptors, and we compared their ability for G protein activation in order to obtain a clue to the roles of the second and third cytoplasmic loops of rhodopsin. The mutants bearing the third loop of four other G(o)-coupled receptors belonging to the rhodopsin superfamily showed significant G(o) activation, indicating that the third loop of rhodopsin possibly has a putative site(s) related to the interaction of G protein and that it is simply exchangeable with those of other G(o)-coupled receptors. The mutants bearing the second loop of other receptors, however, had little ability for G protein activation, suggesting that the second loop of rhodopsin contains a specific region essential for rhodopsin to be a G protein-activating form. Systematic chimeric and point mutational studies indicate that three amino acids (Glu(134), Val(138), and Cys(140)) in the N-terminal region of the second loop of rhodopsin are crucial for efficient G protein activation. These results suggest that the second and third cytoplasmic loops of bovine rhodopsin have distinct roles in G protein activation and subtype specificity.

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.

Binding of arrestin to cytoplasmic loop mutants of bovine rhodopsin.

The binding of arrestin to rhodopsin is a multistep process that begins when arrestin interacts with the phosphorylated C terminus of rhodopsin. This interaction appears to induce a conformational change in arrestin that exposes a high-affinity binding site for rhodopsin. Several studies in which synthetic peptides were used have suggested that sites on the rhodopsin cytoplasmic loops are involved in this interaction. However, the precise amino acids on rhodopsin that participate in this interaction are unknown. This study addresses the role of specific amino acids in the cytoplasmic loops of rhodopsin in binding arrestin through the use of site-directed mutagenesis and direct binding assays. A series of alanine mutants within the three cytoplasmic loops of rhodopsin were expressed in HEK-293 cells, reconstituted with 11-cis-retinal, prephosphorylated with rhodopsin kinase, and examined for their ability to bind in vitro-translated, 35S-labeled arrestin. Mutations at Asn-73 in loop I as well as at Pro-142 and Met-143 in loop II resulted in dramatic decreases in the level of arrestin binding, whereas the level of phosphorylation by rhodopsin kinase was similar to that of wild-type rhodopsin. The results indicate that these amino acids play a significant role in arrestin binding.

Rhodopsin arginine-135 mutants are phosphorylated by rhodopsin kinase and bind arrestin in the absence of 11-cis-retinal.

Arginine-135, located at the border between the third transmembrane domain and the second cytoplasmic loop of rhodopsin, is one of the most highly conserved amino acids in the family of G protein-coupled receptors. The effect of mutation at Arg-135 on the ability of rhodopsin to undergo desensitization was investigated. Four mutants, R135K, R135Q, R135A, and R135L, were examined for their ability to be phosphorylated by rhodopsin kinase, to bind arrestin, and to activate the rod cell G protein, transducin (Gt). All of the mutants were phosphorylated, bound arrestin, and were able to activate Gt when reconstituted with 11-cis-retinal. Surprisingly, several of the mutants could be phosphorylated by rhodopsin kinase and could bind arrestin in the absence of 11-cis-retinal but were not able to activate Gt. These observations represent the first demonstration of a mutant G protein-coupled receptor that assumes a conformation able to interact with its G protein-coupled receptor kinase and arrestin, but not with its G protein, in the absence of ligand.

The first and second cytoplasmic loops of the G-protein receptor, rhodopsin, independently form beta-turns.

The cytoplasmic face of the transmembrane protein, rhodopsin, is made up of one carboxyl terminal and three cytoplasmic loops connecting six of the seven transmembrane helices. Neither the high-resolution, three-dimensional structure of this G-protein receptor nor any other cell surface receptor is known. In this work, the structures of peptides containing the amino acid sequence of the first and second cytoplasmic loops of rhodopsin have been determined. Both loops show ordered structures in solution. In both loops, the ends of the transmembrane helices unwind and form a beta-turn. The conformations of the two loops are remarkably similar, even though their sequences are not. These data suggest a structural motif for short loops in transmembrane proteins. The well-ordered structures of these loops, in the absence of the transmembrane helices, indicate that the primary sequences of these loops stabilize the beta-turn. These data further suggest that the loops may contribute to the folding of such membrane proteins during their synthesis and insertion into membranes.

Structure and function in rhodopsin: peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase.

Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in Vmax for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R/R135E showed a 50% reduction in Vmax relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the Km by 5-fold for E239Q and E247Q/K248L/E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.

Structure of the third cytoplasmic loop of bovine rhodopsin.

The three-dimensional high-resolution structure of rhodopsin is unknown, as is the case for almost all integral membrane patients. As part of an alternative approach to determine of membrane protein structure, we are pursuing the structure of cytoplasmic domains of this G-protein receptor. A peptide, rhoIII, with the sequence of the third cytoplasmic loop of bovine rhodopsin was synthesized. This soluble peptide was biologically active, inhibiting the light-stimulated activation of the rod cell phosphodiesterase by rhodopsin in rod outer segment disks. Therefore rhoIII likely contains structural elements characteristic of native rhodopsin. The structure of rhoIII was determined by H nuclear magnetic resonance. A defined structure was obtained for about 70% of rhoIII. A model of a turn-helix-turn motif could then be proposed for the third cytoplasmic loop of rhodopsin, which suggested a molecular switch for activation of the G-protein by the receptor.

Rhodopsin Mutants Discriminate Sites Important for the Activation of Rhodopsin Kinase and G

The cytoplasmic loops of rhodopsin, the rod cell photoreceptor, play important regulatory roles in the activation of both rhodopsin kinase and the rod cell G protein, Gt. A number of studies have identified domains in rhodopsin that are important for the activation of Gt. However, less is known concerning the cytoplasmic regions that regulate phosphorylation of the photoreceptor by rhodopsin kinase. To identify regions that participate in these processes, a series of alanine mutations were generated in the three cytoplasmic loops of rhodopsin and transiently expressed in HEK-293 cells. Membranes prepared from these cells were reconstituted with the opsin chromophore, 11-cis-retinal, and characterized for their ability to undergo light-dependent phosphorylation by rhodopsin kinase and to catalyze GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to Gt. We have identified mutants that fall into three distinct categories: 1) those that show altered phosphorylation but normal Gt activation, such as T62A/V63A/Q64A and R147A/F148A/G149A in Loops I and II, respectively; 2) mutants that have reduced ability to activate Gt but are phosphorylated normally, including T242A/T243A and V250A/T251A/R252A in Loop III; and 3) mutants that affect both phosphorylation and Gt activation, including A233G/A234G/A235G and A233N/A234N/A235N in Loop III. The use of these two assays in parallel have allowed us to distinguish the presence of distinct functional domains within the cytoplasmic loops which are specific for interaction with rhodopsin kinase or Gt.

Rhodopsin Mutants That Bind But Fail to Activate Transducin

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.

Labelling of the cytoplasmic domains of ovine rhodopsin with hydrophilic chemical probes

The disposition of polypeptide chain of ovine rhodopsin in the photoreceptor disc membrane was investigated by using two hydrophilic reagents, 3,5-di-[125I]iodo-4-diazobenzenesulphonate [( 125I]DDISA) and [14C]succinic anhydride. Both reagents were used to modify rhodopsin in intact disc membranes under conditions where no loss of A500 occurred. Reaction of [125I]DDISA with rhodopsin approached completion after 30 min. Binding was saturated at a 75-fold molar excess of reagent, which gave binding ratios of up to 2 mol/mol of rhodopsin. Proteolysis of rhodopsin, using Staphylococcus aureus V8 proteinase, yielded two membrane-bound fragments, both of which contained bound radioactive probe. Subsequent CNBr cleavage of these fragments produced five radiolabelled peptides which corresponded to the C-terminal region and cytoplasmic loops of rhodopsin. Similar studies with [14C]-succinic anhydride also gave binding ratios of up to 2 mol/mol of rhodopsin. Sequencing of the [14C]succinylated peptides identified the location of the reactive sites as lysine residues 66, 67, 141, 245, 248, 311, 325 and 339 in the polypeptide chain. Non-permeability of both probes was demonstrated by the absence of any radioactivity associated with the intradiscal N-terminal glycopeptide. Sonication of membranes in the presence of [125I]DDISA led to the incorporation of label in this peptide.