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PubMed2003

The eye of the african mole-rat Cryptomys anselli: to see or not to see?

In an attempt to clarify its possible physiological role, we studied the eye of the Zambian mole rat Cryptomys anselli by light, electron and confocal microscopy using conventional staining as well as immunolabelling with rod and cone cell markers. The small eyes of Cryptomys are located superficially and display all features typical of sighted animals: iris, pupil and well-developed lens, separating the anterior chamber and the vitreous. The retina shows a well stratified organization and the folds described in blind subterranean or nocturnal mammals were not observed. The major population of the photoreceptor cells in the Cryptomys retina consists of rod cells, again with a morphology quite similar to that found in sighted animals. The relatively short outer segments contain numerous well-stacked disks and show a strong rod-opsin as well as transducin immunoreaction. Synapses were evident in the spherules, the round basal processes of the rod cell, but they lacked the precise organization reported for sighted mammals. Cone cells were present as well, as indicated by peanut lectin staining, but no immunolabelling with polyclonal M/L-opsin antisera was detectable. The presence of cone cells was also suggested by some basal processes at the outer plexiform layer which displayed several synaptic active sites and irregular contours. While the other retinal layers also showed an organization typical of sighted mammals, there were signs of less tightly preserved morphology as well. Displaced rods and amacrine and/or ganglion cells were observed, and some sparse rod spherules penetrated into the inner nuclear layer. A major reduction was observed in the number of ganglion cells, estimated from the number of axons in the optic nerve, that was very low (approximately 1000 per retina on average) relative to sighted mammals. The data we have suggest a slow, ongoing loss of cells with ageing. Apoptotic nuclei, mainly corresponding to photoreceptor cells and ganglion cells, were detected in young individuals, and an overall reduction in the thickness of the retina was observed in older animals. The morphological data presented here allow some first speculations on the physiological role of the Cryptomys eye and will hopefully trigger detailed studies on the chronobiology and the anatomy of the retinal projections and of the visual cortex of this remarkable species.

Acute-Phase Proteins↗
PubMed2005

Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways.

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF(beta-TrCP). The F-box protein beta-TrCP (beta-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to beta-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved beta-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in beta-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to beta-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of beta-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second beta-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.

Amino Acid Sequence↗
PubMed1986

Identification of the probable site of choleragen-catalyzed ADP-ribosylation in a Go alpha-like protein based on cDNA sequence.

Go alpha, a 39-kDa guanyl nucleotide-binding protein, is functionally and structurally similar to the alpha subunits of the stimulatory and inhibitory guanyl nucleotide-binding proteins (Gs alpha, Gi alpha) of adenylate cyclase and to the alpha subunit of transducin (T alpha), the guanyl nucleotide-binding protein of the retinal photon reception system. A cDNA clone was isolated from a bovine retinal lambda gt10 library by using oligonucleotide probes complementary to sequences in two putative T alpha clones. Partial sequence analysis revealed a deduced amino acid sequence identical to sequences of four tryptic peptides from bovine brain Go alpha. Gs alpha and T alpha are known to serve as substrates for ADP-ribosylation by choleragen. Other workers have established the sequence of the tetrapeptide in T alpha containing the arginine that is ADP-ribosylated and its location in the amino acid sequence deduced from T alpha cDNA. The Go alpha cDNA described here includes a region encoding an amino acid sequence very similar to that surrounding the ADP-ribosylation site in T alpha, consistent with observations that Go alpha can also be a substrate for choleragen. A corresponding sequence in the recently identified Gs alpha cDNA is less homologous to that in T alpha or Go alpha. The reported differences in conditions that promote choleragen-catalyzed ADP-ribosylation of Gs alpha vs. Go alpha could be related to differences in amino acid sequence in the region of the acceptor arginine.

Adenosine Diphosphate Ribose↗
PubMed1986

Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.

Antisera were raised against purified subunits of regulatory GTP-binding proteins (G proteins) and against synthetic peptides that correspond to defined regions of G proteins. Peptide antisera were generated that recognized all alpha or all beta subunits from Gs, Gi, Go, and transducin; others recognized only Gs alpha or Go alpha. Such cross-reaction or complete specificity for a given alpha subunit was not obtained when purified subunits were injected. Peptide antisera were used to identify G protein subunits in selected tissue membrane preparations by immunoblots.

Animals↗
PubMed1988

Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin.

To investigate the role of different cysteine residues in bovine rhodopsin, a series of mutants were prepared in which the cysteine residues were systematically replaced by serines. The mutant genes were expressed in monkey kidney cells (COS-1) and the mutant opsins were evaluated for their levels of expression, glycosylation patterns, and ability to form the chromophore characteristic of rhodopsin and to activate transducin. Substitution of the three cytoplasmic cysteines (Cys-316, Cys-322, and Cys-323) and the four membrane-embedded cysteines (Cys-140, Cys-167, Cys-222, and Cys-264) produced proteins with wild-type phenotype. Also, single substitutions of Cys-185 gave rise to a wild-type phenotype. In contrast, substitution of the three intradiscal cysteines (Cys-110, Cys-185, and Cys-187) or single substitution of Cys-110 or Cys-187 gave proteins that were expressed at reduced levels, glycosylated abnormally, and unable to bind 11-cis-retinal. Thus, of the 10 cysteines in bovine rhodopsin, only intradiscal Cys-110 and Cys-187 are essential for the correct tertiary structure of the protein.

Amino Acid Sequence↗
PubMed1990

Role of the intradiscal domain in rhodopsin assembly and function.

The role of the intradiscal polypeptide loops in bovine rhodopsin has been investigated by deletions in the N-terminal tail and in loops B-C, D-E, and E-F as well as by single amino acid substitutions in the D-E loop. Mutants with three types of phenotypes were observed. Type I mutants showed a rhodopsin-like chromophore and glycosylation. Type II mutants did not regenerate the chromophore and showed abnormal glycosylation. Type III mutants showed poor chromophore regeneration and abnormal glycosylation. Reduced transducin activation was shown by some type I and III mutants. Single amino acid substitutions in the D-E loop gave mostly type I mutants. Deletions in loops B-C, D-E, and F-G gave type II mutants, whereas deletions in the N-terminal tail produced type III mutants. Systematic deletions of two adjacent amino acids in loop D-E indicated that the amino acid sequences 171-182 and 189-192 were essential to rhodopsin structure. Immunofluorescence double-staining and transmission electron microscopy of one type II mutant (with residues 189 and 190 deleted) showed that it was mostly in the endoplasmic reticulum, whereas the wild-type protein was in the plasma membrane. We conclude that the first step in the assembly of the rhodopsin molecule is the formation of a three-dimensional structure in the intradiscal domain involving the bulk of the out-of-the-membrane polypeptide segments followed by the linkage of Cys-110 and Cys-187 through a disulfide bond.

Amino Acid Sequence↗
PubMed1991

Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors.

An inhibitory subunit (P gamma) of cGMP phosphodiesterase from vertebrate rod photoreceptors (frog, toad, and bovine) was phosphorylated by cytosolic protein kinase(s) derived from intact frog rod outer segments. The phosphorylation of frog P gamma was stimulated by phosphatidylinositol but not by cAMP or cGMP. One- and two-dimensional gel electrophoresis revealed that 70-80% of P gamma was phosphorylated with 1 mol of phosphate per frog P gamma under optimal conditions. A peptide that derived from an active domain of bovine P gamma was also phosphorylated. Phosphorylation of frog P gamma was inhibited by addition of the peptide to the reaction mixture. Phosphorylation of frog P gamma was also inhibited by addition of transducin subunits or active (P gamma-less) cGMP phosphodiesterase. Okadaic acid, on the other hand, enhanced P gamma phosphorylation, suggesting the presence of protein phosphatase(s) in the cytosolic fraction. These data suggest another mechanism for the regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

In vivo assembly of rhodopsin from expressed polypeptide fragments.

Rhodopsin folding and assembly were investigated by expression of five bovine opsin gene fragments separated at points corresponding to proteolytic cleavage sites in the second or third cytoplasmic regions. The CH(1-146) and CH(147-348) gene fragments encode amino acids 1-146 and 147-348 of opsin, while the TH(1-240) and TH(241-348) gene fragments encode amino acids 1-240 and 241-348, respectively. Another gene fragment, CT(147-240), encodes amino acids 147-240. All five opsin polypeptide fragments were stably produced upon expression of the corresponding gene fragments in COS-1 cells. The singly expressed polypeptide fragments failed to form a chromophore with 11-cis-retinal, whereas coexpression of two or three complementary fragments [CH(1-146) + CH(147-348), TH(1-240) + TH(241-348), or CH(1-146) + CT(147-240) + TH(241-348)] formed pigments with spectral properties similar to wild-type rhodopsin. The NH2-terminal polypeptide in these rhodopsins showed a glycosylation pattern characteristic of wild-type COS-1 cell rhodopsin and was noncovalently associated with its complementary fragment(s). Further, the CH(1-146) + CH(147-348) rhodopsin showed substantial light-dependent activation of transducin. We conclude that the functional assembly of rhodopsin is mediated by the association of at least three protein-folding domains.

Animals↗
PubMed1996

How photons start vision.

Recent studies have elucidated how the absorption of a photon in a rod or cone cell leads to the generation of the amplified neural signal that is transmitted to higher-order visual neurons. Photoexcited visual pigment activates the GTP-binding protein transducin, which in turn stimulates cGMP phosphodiesterase. This enzyme hydrolyzes cGMP, allowing cGMP-gated cationic channels in the surface membrane to close, hyperpolarize the cell, and modulate transmitter release at the synaptic terminal. The kinetics of reactions in the cGMP cascade limit the temporal resolution of the visual system as a whole, while statistical fluctuations in the reactions limit the reliability of detection of dim light. Much interest now focuses on the processes that terminate the light response and dynamically regulate amplification in the cascade, causing the single photon response to be reproducible and allowing the cell to adapt in background light. A light-induced fall in the internal free Ca2+ concentration coordinates negative feedback control of amplification. The fall in Ca2+ stimulates resynthesis of cGMP, antagonizes rhodopsin's catalytic activity, and increases the affinity of the light-regulated cationic channel for cGMP. We are using physiological methods to study the molecular mechanisms that terminate the flash response and mediate adaptation. One approach is to observe transduction in truncated, dialyzed photoreceptor cells whose internal Ca2+ and nucleotide concentrations are under experimental control and to which exogenous proteins can be added. Another approach is to observe transduction in transgenic mouse rods in which specific proteins within the cascade are altered or deleted.

Animals↗
PubMed1998

Light-activated rhodopsin induces structural binding motif in G protein alpha subunit.

A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these receptors recognize and promote nucleotide exchange on G protein alpha subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) alpha subunit C-terminal undecapeptide Gtalpha(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 Gtalpha(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 alphaL 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.

Amino Acid Sequence↗
PubMed1996

Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments). Evidence for ubiquitinylation of Gt and rhodopsin.

In corroboration of the hypothesized regulation of phototransduction proteins by the ubiquitin-dependent pathway, we identified free ubiquitin (8 kDa) and ubiquitin-protein conjugates (50 to >200 kDa; pI 5.3-6.8 by two-dimensional electrophoresis) in bovine rod outer segments (ROS). A 38-kDa ubiquitinylated protein and transducin (Gt) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and Gt were readily solubilized in buffer lacking GTP. These data are consistent with ubiquitinylation of Gt and corroborate previous cell-free experiments identifying Gt as a substrate for ubiquitin-dependent proteolysis (Obin, M. S., Nowell, T., and Taylor, A. (1994) Biochem. Biophys. Res. Commun. 200, 1169-1176). Evidence for ubiquitinylation of rhodopsin (36 kDa), the (photo)receptor coupled to Gt, included (i) the presence in ROS membranes "stripped" of peripheral membrane proteins of numerous ubiquitin-protein conjugates, including two whose masses (44 and 50 kDa) are consistent with mono- and diubiquitinylated rhodopsin; (ii) catalysis by permeabilized ROS of 125I-labeled ubiquitin-protein conjugates whose masses (42, 50, and 58 kDa) suggest a "ladder" of mono-, di-, and triubiquitinylated rhodopsin; (iii) parallel mobility shifts on SDS-polyacrylamide gels of rhodopsin and these 125I-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of 125I-labeled ubiquitin-protein conjugates when stripped, detergent-solubilized ROS membranes (95% rhodopsin) were incubated with reticulocyte lysate. A functional ubiquitin-dependent pathway in ROS is demonstrated by the presence of (i) the ubiquitin-activating enzyme (E1); (ii) four ubiquitin carrier proteins (E214K, E220K, E225K, and E235K) and pronounced activity of E214K, an enzyme required for "N-end rule" proteolysis; (iii) ATP-dependent 26 S proteasome activity that rapidly degrades high mass 125I-labeled ubiquitin-ROS protein conjugates; and (iv) distinct ubiquitin C-terminal isopeptidase/hydrolase activities, including potent ubiquitin-aldehyde-insensitive activity directed at high mass ubiquitinylated moieties. Considered together, the data support a novel role for the ubiquitin-dependent pathway in the regulation of mammalian phototransduction protein levels and/or activities and provide the first identification of a non-calpain proteolytic system in photoreceptors.

Animals↗
PubMed1996

Functional interaction of transmembrane helices 3 and 6 in rhodopsin. Replacement of phenylalanine 261 by alanine causes reversion of phenotype of a glycine 121 replacement mutant.

Replacement of a highly conserved glycine residue on transmembrane (TM) helix 3 of bovine rhodopsin (Gly121) by amino acid residues with larger side chains causes a progressive blue-shift in the lambdamax value of the pigment, a decrease in thermal stability, and an increase in reactivity with hydroxylamine. In addition, mutation of Gly121 causes a relative reversal in the selectivity of opsin for 11-cis-retinal over all-trans-retinal. It was suggested that Gly121 plays an important role in defining the 11-cis-retinal binding pocket of rhodopsin (Han, M., Lin, S. W., Smith, S. O., and Sakmar, T. P. (1996) J. Biol. Chem. 271, 32330-32336). Here, we combined the mutant opsin G121L with second site replacements of four different amino acid residues on TM helix 6: Met257, Val258, Phe261, or Trp265. We show that the loss of function phenotypes of the G121L mutant described above can be partially reverted specifically by the mutation of Phe261, a residue highly conserved in all G protein-coupled receptors. For example, the double-replacement mutant G121L/F261A has spectral, chromophore-binding, and transducin-activating properties intermediate between those of G121L and rhodopsin. This rescue of the G121L defects did not occur with the other second site mutations tested. We conclude that specific portions of TM helices 3 and 6, which include Gly121 and Phe261, respectively, define the chromophore-binding pocket in rhodopsin. Finally, the results are placed in the context of a molecular graphics model of the TM domain of rhodopsin, which includes the retinal-binding pocket.

Alanine↗
PubMed1997

Molecular diversity of adenylyl cyclases in human and rat myometrium. Correlation with global adenylyl cyclase activity during mid- and term pregnancy.

Expression and regulation of myometrial adenylyl cyclases (AC) were studied during pregnancy. Hybridization of poly(A)+ RNA with specific cDNA probes for enzyme types I-IX indicated 1) the presence of transcripts encoding types II-VI and type IX in rat and human, and type VII in rat and 2) the absence of detectable mRNA for types I and VIII in both species. No substantial change was observed in the amount of specific mRNA and basal AC activity from mid-pregnancy to term. However, activation of the alpha2-adrenergic receptor/Gi protein pathway resulted in potentiation of Gs-stimulated AC activity at mid-pregnancy but not at term (Mhaouty, S., Cohen-Tannoudji, J., Bouet-Alard, R., Limon-Boulez, I., Maltier, J. P., and Legrand, C. (1995) J. Biol. Chem. 270, 11012-11016). We demonstrate in the present work that betagamma scavengers transducin-alpha and QEHA peptide abolished this positive input. On the other hand, increasing submicromolar concentrations of free Ca2+, a situation that mimics late term, reduced the forskolin-stimulated AC activity with an IC50 of 3.9 microM. Thus, the presence in myometrium of AC II family (types II, IV, VII) confers ability to G inhibitory proteins to stimulate enzyme activity via betagamma complexes at mid-pregnancy, whereas expression of AC III, V, and VI isoforms confers to the myometrial AC system a high sensitivity to inhibition by Ca2+-dependent processes at term. These data suggest that in the pregnant myometrium, the expression of different species of AC with distinct regulatory properties provides a mechanism for integrating positively or negatively the responses to various hormonal inputs existing either during pregnancy or in late term.

Adenylyl Cyclases↗
PubMed1997

Real time conformational changes in the retinal phosphodiesterase gamma subunit monitored by resonance energy transfer.

The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT). In order to characterize conformational changes in the 87-amino acid gammaPDE subunit that may accompany the activation of the holoenzyme, gammaPDE was labeled with the fluorescent probes 5-iodoacetamidofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements. 5-Iodoacetamidofluorescein specifically labeled a cysteine residue at position 68 and served as a resonance energy transfer donor. The site of modification of eosin-5-isothiocyanate, which served as the resonance energy transfer acceptor, was determined to be within the first seven residues of the amino terminus of gammaPDE. Energy transfer between the labeled sites on free, unbound gammaPDE indicated that they were separated by a distance of 63 A, consistent with a random conformation. Upon binding the catalytic alphabeta subunits of the PDE, the distance between the two probes on gammaPDE increased to 77 A. Binding of the labeled gammaPDE by alphaT.guanosine 5'-3-O-(thio)triphosphate did not affect the distance between the probes under conditions where the PDE was activated. These data are consistent with the view that the binding of activated alphaT to gammaPDE, which is essential for the stimulation of PDE activity, does not impart significant alterations in the tertiary structure of the gammaPDE molecule. They also support a model for PDE activation that places active alphaT in a complex with the holoenzyme.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9.

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

beta-TrCP mediates the signal-induced ubiquitination of IkappaBbeta.

We have examined the role of beta-TrCP (beta-transducin repeat-containing protein) in the ubiquitination and degradation of IkappaBbeta, one of the two major IkappaB isoforms in mammalian cells. We demonstrate that beta-TrCP interacts specifically with IkappaBbeta, and such interaction is dependent on prior phosphorylation of IkappaBbeta on serines 19 and 23. Interaction with beta-TrCP is also necessary for ubiquitination of IkappaBbeta upon stimulation of cells, and deletion of the F-box in beta-TrCP abolishes its ability to ubiquitinate IkappaBbeta. Therefore, these results indicate that beta-TrCP plays a critical role in the activation of NF-kappaB by assembling the ubiquitin ligase complex for both phosphorylated IkappaBalpha and IkappaBbeta.

Animals↗
PubMed2000

Diffusible ligand all-trans-retinal activates opsin via a palmitoylation-dependent mechanism.

In rhodopsin's function as a photoreceptor, 11-cis-retinal is covalently bound to Lys(296) via a protonated Schiff base. 11-cis/all-trans photoisomerization and relaxation through intermediates lead to the metarhodopsin II photoproduct, which couples to transducin (G(t)). Here we have analyzed a different signaling state that arises from noncovalent binding of all-trans-retinal (atr) to the aporeceptor opsin and enhances the very low opsin activity by several orders of magnitude. Like with metarhodopsin II, coupling of G(t) to opsin-atr is sensitive to competition by synthetic peptides from the COOH termini of both G(t)alpha and G(t)gamma. However, atr does not compete with 11-cis-retinal incorporation into the Lys(296) binding site and formation of the light-sensitive pigment. Blue light illumination fails to photorevert opsin-atr to the ground state. Thus noncovalently bound atr has no access to the light-dependent binding site and reaction pathway. Moreover, in contrast to light-dependent signaling, removal of the palmitoyl anchors at Cys(322) and Cys(323) in the rhodopsin COOH terminus impairs the atr-stimulated activity. Repalmitoylation by autoacylation with palmitoyl-coenzyme A restores most of the original activity. We hypothesize that the palmitoyl moieties are part of a second binding pocket for the chromophore, mediating hydrophobic interactions that can activate a large part of the catalytic receptor/G-protein interface.

Acylation↗
PubMed2000

Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches.

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 light-induced 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.

Animals↗