Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transducin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources
PubMed1986

Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs.

Retinal transducin, a guanine nucleotide regulatory protein (referred to as a G protein) that activates a cGMP phosphodiesterase in photoreceptor cells, is comprised of three subunits. We have identified and analyzed cDNA clones of the bovine transducin beta subunit that may be highly conserved or identical to that in other G proteins. From the cDNA nucleotide sequence of the entire coding region, the primary structure of a 340-amino acid protein was deduced. The encoded beta subunit has a Mr of 37,375 and is comprised of repetitive homologous segments arranged in tandem. Furthermore, significant homology in primary structure and segmental sequence exists between the beta subunit and the yeast CDC4 gene product. The Mr 37,375 beta subunit polypeptide is encoded by a 2.9-kilobase (kb) mRNA. However, there exists in retina other beta-related mRNAs that are divergent from the 2.9-kb mRNA on the basis of oligonucleotide and primer-extended probe hybridizations. All mammalian tissues and clonal cell lines that have been examined contain at least two beta-related mRNAs, usually 1.8 and 2.9 kb in length. These results suggest that the mRNAs are the processed products of a small number of closely related genes or of a single highly complex beta gene.

Amino Acid Sequence↗
PubMed1996

High affinity interactions of GTPgammaS with the heterotrimeric G protein, transducin. Evidence at high and low protein concentrations.

A well known difference in nucleotide binding characteristics between heterotrimeric G proteins and small GTP binding proteins of the Ras superfamily is that the former bind GTP or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) with a much lower affinity (Kd approximately 10(-8)-10(-7) M) than the latter (Kd approximately 10(-11)-10(-10)M). We report here that the alpha subunit of the heterotrimeric G protein transducin (Gt) binds GTPgammaS with an affinity comparable to that of Ras. High affinity binding was suggested by GTPgammaS titrations of rod outer segment samples with Gt concentrations in the range of 7 nM to 300 nM; the results were more consistent with a dissociation constant for GTPgammaS in the subnanomolar range, than with one in the 10(-8)-10(-7) M range typically reported for heterotrimeric G proteins. Equilibrium binding experiments with G protein concentrations in the subnanomolar to nanomolar range confirmed this conclusion and revealed a dissociation constant of 50 pM. Thus, transducin's affinity for GTPgammaS, and by inference, for GTP, appears to be approximately three orders of magnitude higher than previously reported. These results raise the possibility that some results obtained with high concentrations of nucleotide analogues may be due to minute traces of contaminants such as GTP, GTPgammaS, or GTPalphaS, that have high affinities for Gtalpha.

Guanosine 5'-O-(3-Thiotriphosphate)↗
PubMed1997

The retinal specific protein RGS-r competes with the gamma subunit of cGMP phosphodiesterase for the alpha subunit of transducin and facilitates signal termination.

In vertebrate photoreceptor cells, transducin mediates signaling between rhodopsin and cGMP phosphodiesterase by transiently binding its gamma subunit (PDEgamma). For the termination of signaling GTP hydrolysis by the transducin alpha subunit (TDalpha) GTPase is required. This reaction can be accelerated by GTPase-activating proteins (GAPs), e.g. PDEgamma. Recently we identified a second retinal GAP that interacts with TDalpha, RGS-r. Here we compare the GAP function of RGS-r and PDEgamma. Both proteins stimulated single turnover GTPase of TDalpha; however, RGS-r was more effective than PDEgamma. When added together, PDEgamma competitively inhibited the RGS-r-stimulated GTPase. In addition, the interaction of TDalpha in its GTP-bound form (TDalphaGTPgammaS), the transition state (TDalphaGDP*AMF) and the GDP-bound form (TDalphaGDP) with RGS-r and PDE, respectively, was measured by surface plasmon resonance. PDEgamma displayed highest affinity for TDalphaGTPgammaS, weaker affinity for TDalphaGDP*AMF, and weakest affinity for TDalphaGDP. RGS-r exhibited only a comparable high affinity for TDalphaGDP*AMF. We conclude that the observed competition between RGS-r and PDEgamma for TDalpha occurs when the hydrolysis of GTP is initiated. By competing with PDEgamma and removing it from TDalpha as well as increasing Pi release, RGS-r apparently facilitates signal termination and TDalpha recycling.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Communication between switch II and switch III of the transducin alpha subunit is essential for target activation.

Comparisons of the tertiary structures of the GDP-bound and guanosine 5'-O-(thiotriphosphate) (GTPgammaS)-bound forms of the alpha subunit of transducin (alphaT) indicate that there are three regions that undergo changes in conformation upon alphaT activation. Two of these regions, Switch I and Switch II, were originally identified in Ras, while Switch III appears to be unique to trimeric GTP-binding proteins (G proteins). We find that replacement of the Switch III region (aspartic acid 227 through asparagine 237) with a single alanine residue yields an alphaT subunit that fully binds and hydrolyzes GTP but no longer stimulates the activity of the cyclic GMP phosphodiesterase (PDE), the physiological target for transducin. We also show that changing glutamic acid 232 of alphaT to a leucine (E232L) had no effect on rhodopsin-stimulated GTP-GDP exchange nor on the GTP hydrolytic activity of alphaT. However, the GTPgammaS-bound form of the alphaTE232L mutant was unable to stimulate the activity of the cyclic GMP PDE. The lack of stimulation was not due to an inability of the alphaTE232L mutant to bind to the target. Taken together, these results indicate that glutamic acid 232 mediates a conformational coupling between Switch II and Switch III, which is essential for converting GTP-dependent G protein-target interactions into a stimulation of target/effector activity.

Animals↗
PubMed1998

Identification of effector residues on photoreceptor G protein, transducin.

Transducin is a photoreceptor-specific heterotrimeric GTP-binding protein that plays a key role in the vertebrate visual transduction cascade. Here, using scanning site-directed mutagenesis of the chimeric Galphat/Galphai1 alpha-subunit (Galphat/i), we identified Galphat residues critical for interaction with the effector enzyme, rod cGMP phosphodiesterase (PDE). Our evidence suggests that residue Ile208 in the switch II region directly interacts with the effector in the active GTP-bound conformation of Galphat. Residues Arg201, Arg204, and Trp207 are essential for the conformation-dependent Galphat/effector interaction either via direct contacts with the inhibitory PDE gamma-subunit or by forming an effector-competent conformation through the communication network between switch II and the switch III/alpha3-helix domain of Galphat. Residues His244 and Asn247 in the alpha3 helix of Galphat are responsible for the conformation-independent effector-specific interaction. Insertion of these residues rendered the Galphat/i chimera with the ability to bind PDE gamma-subunit and stimulate PDE activity approaching that of native Galphat. Comparative analysis of the interactions of Galphat/i mutants with PDE and RGS16 revealed two adjacent but distinct interfaces on transducin. This indicates a possibility for a functional trimeric complex, RGS/Galpha/effector, that may play a central role in turn-off mechanisms of G protein signaling systems, particularly in phototransduction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Mechanism of allosteric regulation of the rod cGMP phosphodiesterase activity by the helical domain of transducin alpha subunit.

The G protein alpha subunit (Galpha) is composed of two distinct folding domains: a GTP-binding Ras-like domain and an alpha helical domain (HD). We have recently reported that the helical domain (HDt) of the vertebrate visual transducin alpha subunit (Galphat) synergizes activation of retinal cyclic GMP phosphodiesterase (PDE) by activated Galphat (Liu, W., and Northup, J. K., (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12878-12883). Here, we examine the molecular basis for this HD-based signaling regulation, and we provide a new model for the activation of the target effector. The HD proteins derived from visual transducin or taste gustducin alpha subunits, but no other Galpha HD proteins, each attenuate the PDE catalytic core (Palphabeta) and synergize Galphat stimulation of the holoPDE (Palphabetagamma2) with similar apparent affinities. The data from studies of both HDt-mediated attenuation and stimulation indicate that the HDt and the PDE inhibitory subunit (Pgamma) interact with PDE at independent sites and that Palphabeta contains the binding sites for HD. The saturation of both processes by HDt displays positive cooperativity with Hill coefficients of 1.5 for the attenuation of Palphabeta activity and 2.1 for synergism of holoPDE activation. Our data suggest the that Galphat-HDt regulates PDE by allosterically decreasing the affinity of Palphabeta for Pgamma and thus simultaneously facilitating the interaction of the activated Galphat-Ras-like domain with Pgamma. Thus, we propose a new model for the high efficiency of PDE activation as well as deactivation, and, overall, a novel mechanism for controlling fidelity, sensitivity, and efficacy of G protein signaling.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Rhodopsin recognition by mutant G(s)alpha containing C-terminal residues of transducin.

The C-terminal regions of the heterotrimeric G protein alpha-subunits play key roles in selective activation of G proteins by their cognate receptors. In this study, mutant G(s)alpha proteins with substitutions by C-terminal residues of transducin (G(t)alpha) were analyzed for their interaction with light-activated rhodopsin (R*) to delineate the critical determinants of the G(t)alpha/R* coupling. In contrast to G(s)alpha, a chimeric G(s)alpha/G(t)alpha protein containing only 11 C-terminal residues from transducin was capable of binding to and being potently activated by R*. Our results suggest that Cys(347) and Gly(348) are absolutely essential, whereas Asp(346) is more modestly involved in the G(t) activation by R*. In addition, the analysis of the intrinsic nucleotide exchange in mutant G(s)alpha indicated an interaction between the C terminus and the switch II region in G(t)alpha.GDP. Mutant G(s)alpha containing the G(t)alpha C terminus and substitutions of Asn(239) and Asp(240) (switch II) by the corresponding G(t)alpha residues, Glu(212) and Gly(213), displayed significant reductions in spontaneous guanosine 5'-O-(3-thiotriphosphate)-binding rates to the levels approaching those in G(t)alpha. Communication between the C terminus and switch II of G(t)alpha does not appear essential for the activational coupling between G(t) and R*, but may represent one of the mechanisms by which Galpha subunits control intrinsic nucleotide exchange.

Amino Acid Sequence↗
PubMed2000

Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin.

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.

Amino Acid Sequence↗
PubMed2000

AGS3 inhibits GDP dissociation from galpha subunits of the Gi family and rhodopsin-dependent activation of transducin.

A number of recently discovered proteins that interact with the alpha subunits of G(i)-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs. Activator of G protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats. To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by G(i)alpha(1) and transducin-alpha (G(t)alpha). The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to G(i)alpha and rhodopsin-stimulated GTPgammaS binding to G(t)alpha. The full-length AGS3 GPR domain, AGS3-(463-650), was approximately 30-fold more potent than AGS3-(572-629), containing two AGS3 GPR motifs. The IC(50) values for the AGS3-(463-650) inhibitory effects on G(i)alpha and transducin were 0.12 and 0.15 microm, respectively. Furthermore, AGS3-(463-650) and AGS3-(572-629) effectively blocked the GDP release from G(i)alpha and rhodopsin-induced dissociation of GDP from G(t)alpha. The potencies of AGS3-(572-629) and AGS3-(463-650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTPgammaS binding. Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by G(i)alpha. The catalytic rate of G(t)alpha GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463-650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of Gbetagamma signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor.

Amino Acid Motifs↗
PubMed2002

Ubiquitylation of the transducin betagamma subunit complex. Regulation by phosducin.

G proteins (Galphabetagamma) are essential signaling molecules, which dissociate into Galpha and Gbetagamma upon activation by heptahelical membrane receptors. We have identified the betagamma subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin gamma-subunit (Tgamma) but not the alpha- or beta-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tgamma was exclusively ubiquitylated when Tbetagamma was dissociated from Talpha. Ubiquitylation of Tbetagamma on Tgamma was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tbetagamma subunit complex in vitro by a mechanism requiring ATP and the proteasome. We also show that Tbetagamma association with phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tbetagamma ubiquitylation and subsequent degradation. Phosphorylation of phosducin by Ca(2+)/calmodulin-dependent protein kinase II, which inhibits phosducin-Tbetagamma complex formation, completely restored Tbetagamma ubiquitylation and degradation. We conclude that Tbetagamma is a substrate of the ubiquitin-proteasome pathway and suggest that phosducin serves to protect Tbetagamma following the light-dependent dissociation of Talphabetagamma.

Animals↗
PubMed2004

Perturbing the linker regions of the alpha-subunit of transducin: a new class of constitutively active GTP-binding proteins.

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the alpha subunits of transducin (alpha(T)) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Galpha subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an alpha(T)/alpha(i1) chimera (designated alpha(T)(*)). Both alpha(T)(*) linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal alpha(T) or recombinant alpha(T)(*). The alpha(T)(*) linker mutants were responsive to aluminum fluoride, which binds to alpha-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the betagamma complex, their activation was not influenced by betagamma alone, arguing against the idea that the betagamma complex helps to pry apart the helical and Ras-like domains of Galpha subunits. Once activated, the alpha(T)(*) linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Galpha mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2006

The N terminus of GTP gamma S-activated transducin alpha-subunit interacts with the C terminus of the cGMP phosphodiesterase gamma-subunit.

Dynamic regulation of G-protein signaling in the phototransduction cascade ensures the high temporal resolution of vision. In a key step, the activated alpha-subunit of transducin (Galphat-GTP) activates the cGMP phosphodiesterase (PDE) by binding the inhibitory gamma-subunit (PDEgamma). Significant progress in understanding the interaction between Galphat and PDEgamma was achieved by solving the crystal structure of the PDEgamma C-terminal peptide bound to Galphat in the transition state for GTP hydrolysis (Slep, K. C., Kercher, M. A., He, W., Cowan, C. W., Wensel, T. G., and Sigler, P. B. (2001) Nature 409, 1071-1077). However, some of the structural elements of each molecule were absent in the crystal structure. We have probed the binding surface between the PDEgamma C terminus and activated Galphat bound to guanosine 5'-O-(3-thio)-triphosphate (GTPgammaS) using a series of full-length PDEgamma photoprobes generated by intein-mediated expressed protein ligation. For each of seven PDEgamma photoprobe species, expressed protein ligation allowed one benzoyl-L-phenylalaine substitution at selected hydrophobic C-terminal positions, and the addition of a biotin affinity tag at the extreme C terminus. We have detected photocross-linking from several PDEgamma C-terminal positions to the Galphat-GTPgammaS N terminus, particularly from PDEgamma residue 73. The overall percentage of cross-linking to the Galphat-GTPgammaSN terminus was analyzed using a far Western method for examining Galphat-GTPgammaS proteolytic digestion patterns. Furthermore, mass spectrometric analysis of cross-links to Galphat from a benzoyl-phenylalanine replacement at PDEgamma position 86 localized the region of photoinsertion to Galphat N-terminal residues Galphat-(22-26). This novel Galphat/PDEgamma interaction suggests that the transducin N terminus plays an active role in signal transduction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

Canine rod transducin alpha-1: cloning of the cDNA and evaluation of the gene as a candidate for progressive retinal atrophy.

PURPOSE: Progressive retinal atrophy (PRA) represents a heterogeneous group of retinal dystrophies, distinct forms of which occur in different canine breeds. The present study was undertaken to evaluate the gene for the alpha-1 subunit of the rod specific G-protein transducin (GNAT1), a member of the phototransduction pathway, as a candidate for progressive rod cone degeneration (pred) in poodles, early retinal degeneration (erd) in elkhounds, and rod cone dysplasia 2 (rcd2) in collies. METHODS: Oligonucleotide primers were designed from the consensus region of known cDNA sequences for GNAT1 from other species. Canine GNAT1 cDNA was cloned and sequenced after reverse transcription (RT) and polymerase chain reaction (PCR) of total retinal RNA, and PCR amplification of specific sequences from a canine retinal cDNA library. Large, intron containing fragments of the canine transducin alpha-1 subunit gene were amplified from genomic DNA of individuals in PRA informative pedigrees, using canine-specific primers. PCR products were digested with Nci I, to enable typing of individuals in the PRA affected pedigrees for a previously identified GNAT1 restriction fragment length polymorphism (RFLP). RESULTS: The sequence of canine GNAT1 cDNA is reported (GenBank accession no. U65376). Over the coding region, the canine GNAT1 cDNA sequence presented here shares 92-95% identity with human, bovine and murine sequences. The canine cDNA encodes a polypeptide of 350 amino acids; its theoretical translation is 98-99% identical with the corresponding GNAT1 sequence from each of the other 3 species and it has no unique amino acids. In rcd2 and erd pedigrees informative for both the disease locus and the GNAT1 Nci I RFLP, a minimum of 3 and 2 recombinants were identified, respectively. Similarly, in a prcd pedigree, 3 of 7 progeny informative for both prcd and this RFLP were obligate recombinants. CONCLUSIONS: The canine GNAT1 gene has been excluded as a candidate for prcd, erd and rcd2. Sequence information of canine GNAT1 gene will enable testing this locus as a candidate in other canine hereditary retinal degenerations.

Amino Acid Sequence↗
PubMed1994

Improved large-scale purification of transducin, and its alpha and beta gamma subunits from frozen retinas.

The transducin heterotrimer and its alpha- and beta gamma-subunits have been purified from frozen bovine rod outer segments by modifying existing procedures. The methods described here are relatively simple and fast. The yield (ca. 8 mgs/100 retinas) and purity of the transducin heterotrimer and subunits from frozen retinas is equal to or larger than those previously obtained from fresh or frozen retinas.

Animals↗
PubMed2006

Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange.

The mechanisms by which G-protein-coupled receptors (GPCRs) activate G-proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G-protein complexes. For study of GPCR/G-protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human beta2-adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTPgamma35S/GDP exchange of the transducin alpha subunit (Galphat) at a level 3.5-fold higher than the basal exchange. This activation was inhibited by GDP and by a high-affinity peptide analog of the Galphat C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Galphat C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Galphat-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions.

Animals↗
PubMed2004

Early receptor current of wild-type and transducin knockout mice: photosensitivity and light-induced Ca2+ release.

We have used suction-electrode recording to measure the early receptor current (ERC) from single, isolated mammalian photoreceptors. When a wild-type mouse rod was illuminated with light sufficient to close all the cGMP-gated channels, a succeeding bright laser flash bleaching a large proportion of the visual pigment produced an ERC, which at 37 degrees C consisted primarily of a single component of transient positive current. The amplitude of total charge movement of this component declined exponentially with successive flashes, consistent with the direct proportionality of the ERC to the quantity of pigment bleached. From the constant of exponential decline, it was possible to estimate the in vivo photosensitivity of mouse rhodopsin to be about 6 x 10(-9)microm(2) per molecule. We have also measured the ERC from rods of transducin-knockout mice, for which previous illumination to close the cGMP-gated channels was not required. The ERC of these rods was similar to that of wild-type rods but was followed by a slow component of outward current whose maximum amplitude in some cells approached that of the normal light response. This slow current was blocked by l-cis diltiazem, indicating that it was produced by ion flux through the cyclic nucleotide-gated channels of the outer segment; however, it could not have been produced by the normal transduction cascade, since it was recorded from rods lacking transducin. Since it was depressed by prior incorporation of the Ca(2+) buffer BAPTA, it was probably generated by light-activated Ca(2+) release earlier demonstrated in salamander and zebrafish. Recordings of the ERC from normal and mutant mice may provide a useful tool for the analysis of models of retinal disease, as well as exploration of the molecular origin of light-activated Ca(2+) release.

Animals↗
PubMed1991

Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore.

Rhodopsin and the visual pigments are a distinct group within the family of G-protein-linked receptors in that they have a covalently bound ligand, the 11-cis-retinal chromophore, whereas all of the other receptors bind their agonists through noncovalent interactions. The retinal chromophore in rhodopsin is bound by means of a protonated Schiff base linkage to the epsilon-amino group of Lys-296. Two rhodopsin mutants have been constructed, K296G and K296A, in which the covalent linkage to the chromophore is removed. Both mutants form a pigment with an absorption spectrum close to that of the wild type when reconstituted with the Schiff base of an n-alkylamine and 11-cis-retinal. In addition, the pigment formed from K296G and the n-propylamine Schiff base of 11-cis-retinal was found to activate transducin in a light-dependent manner, with 30 to 40% of the specific activity measured for the wild-type protein. It appears that the covalent bond is not essential for binding of the chromophore or for catalytic activation of transducin.

Binding Sites↗
PubMed1996

Uncoupling of GTP binding from target stimulation by a single mutation in the transducin alpha subunit.

Glutamic acid-203 of the alpha subunit of transducin (alphaT) resides within a domain that undergoes a guanosine triphosphate (GTP)-induced conformational change that is essential for effector recognition. Changing the glutamic acid to an alanine in bovine alpha(T) yielded an alpha subunit (alpha(T)E203A) that was fully dependent on rhodopsin for GTP-guanosine diphosphate (GDP) exchange and showed GTP hydrolytic activity similar to that measured for wild-type alpha(T). However, unlike the wild-type protein, the GDP-bound form of alpha(T)E203A was constitutively active toward the effector of transducin, the cyclic guanosine monophosphate phosphodiesterase. Thus, the alpha(T)E203A mutant represents a short-circuited protein switch that no longer requires GTP for the activation of the effector target phosphodiesterase.

3',5'-Cyclic-GMP Phosphodiesterases↗