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PubMed1999

Ggamma13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium.

Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The alpha subunit of gustducin (alpha-gustducin) is critical for transduction of responses to bitter or sweet compounds. We identified a G-protein gamma subunit (Ggamma13) that colocalized with alpha-gustducin in taste receptor cells. Of 19 alpha-gustducin/Ggamma13-positive taste receptor cells profiled, all expressed the G protein beta3 subunit (Gbeta3); approximately 80% also expressed Gbeta1. Gustducin heterotrimers (alpha-gustducin/Gbeta1/Ggamma13) were activated by taste cell membranes plus bitter denatonium. Antibodies against Ggamma13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducin heterotrimers transduce responses to bitter and sweet compounds via alpha-gustducin's regulation of phosphodiesterase (PDE) and Gbetagamma's activation of phospholipase C (PLC).

Amino Acid Sequence↗
PubMed2000

Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1.

Timely deactivation of the alpha-subunit of the rod G-protein transducin (Galphat) is essential for the temporal resolution of rod vision. Regulators of G-protein signalling (RGS) proteins accelerate hydrolysis of GTP by the alpha-subunits of heterotrimeric G proteins in vitro. Several retinal RGS proteins can act in vitro as GTPase accelerating proteins (GAP) for Galphat. Recent reconstitution experiments indicate that one of these, RGS9-1, may account for much of the Galphat GAP activity in rod outer segments (ROS). Here we report that ROS membranes from mice lacking RGS9-1 hydrolyse GTP more slowly than ROS membranes from control mice. The Gbeta5-L protein that forms a complex with RGS9-1 was absent from RGS9-/- retinas, although Gbeta5-L messenger RNA was still present. The flash responses of RGS9-/- rods rose normally, but recovered much more slowly than normal. We conclude that RGS9-1, probably in a complex with Gbeta5-L, is essential for acceleration of hydrolysis of GTP by Galphat and for normal recovery of the photoresponse.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

NMR structure of a receptor-bound G-protein peptide.

Heterotrimeric GTP-binding proteins (G proteins) regulate cellular activity by coupling to hormone or sensory receptors. Stimulated receptors catalyse the release of GDP from G protein alpha-subunits and GTP bound to the empty alpha-subunits provides signals that control effectors such as adenylyl cyclases, phosphodiesterases, phospholipases and ion channels. Three cytoplasmic loops of the activated receptor are thought to interact with three sites on the heterotrimeric G protein to provide high-affinity interaction and catalyse G-protein activation. The carboxyl terminus of the alpha-subunit is particularly important for interaction with the receptor. Here we study the structure of part of the active interface between the photon receptor rhodopsin and the G protein transducin, or Gt, using nuclear magnetic resonance. An 11-amino-acid peptide from the C terminus of the alpha-subunit of Gt (alpha t (340-350)) binds to rhodopsin and mimics the G protein in stabilizing its active form, metarhodopsin II. The peptide alpha t (340-350) binds to both excited and unexcited rhodopsin and conformational differences between the two bound forms suggest a mechanism for activation of G proteins by agonist-stimulated receptors. Insight into receptor-catalysed GDP release will have broad application because the GTP/GDP exchange and the intrinsic GTPase activity of GTP-binding proteins constitute a widespread regulatory mechanism.

Amino Acid Sequence↗
PubMed1994

Structural determinants for activation of the alpha-subunit of a heterotrimeric G protein.

The 1.8 A crystal structure of transducin alpha.GDP, when compared to that of the activated complex with GTP-gamma S, reveals the nature of the conformational changes that occur on activation of a heterotrimeric G-protein alpha-subunit. Structural changes initiated by direct contacts with the terminal phosphate of GTP propagate to regions that have been implicated in effector activation. The changes are distinct from those observed in other members of the GTPase superfamily.

Amino Acid Sequence↗
PubMed1996

Crystal structure of a G-protein beta gamma dimer at 2.1A resolution.

Many signalling cascades use seven-helical transmembrane receptors coupled to heterotrimeric G proteins (G alpha beta gamma) to convert extracellular signals into intracellular responses. Upon nucleotide exchange catalysed by activated receptors, heterotrimers dissociate into GTP-bound G alpha subunits and G beta gamma dimers, either of which can modulate many downstream effectors. Here we use multiwavelength anomalous diffraction data to solve the crystal structure of the beta gamma dimer of the G protein transducin. The beta-subunit is primarily a seven-bladed beta-propeller that is partially encircled by an extended gamma-subunit. The beta-propeller, which contains seven structurally similar WD repeats, defines the stereochemistry of the WD repeat and the probable architecture of all WD-repeat-containing domains. The structure details interactions between G protein beta- and gamma-subunits and highlights regions implicated in effector modulation for the conserved family of G protein beta gamma dimers.

Amino Acid Sequence↗
PubMed1999

The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of beta-catenin.

Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., beta-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Specific F-box proteins (Fbps) recruit different substrates to the SCF. Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported. We have found that one human Fbp, beta-Trcp (beta-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1. Consistent with recent reports indicating that Xenopus and Drosophila beta-Trcp homologs act as negative regulators of the Wnt/beta-catenin signaling pathway, we report here that human beta-Trcp interacts with beta-catenin in vivo. Furthermore, beta-catenin is specifically stabilized in vivo by the expression of a dominant negative beta-Trcp. These results indicate that the Cul1/Skp1/beta-Trcp complex forms a ubiquitin ligase that mediates the degradation of beta-catenin.

CD4 Antigens↗
PubMed2004

The many faces of beta-TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer.

Beta-transducin repeats-containing proteins (beta-TrCP) serve as the substrate recognition subunits for the SCFbeta-TrCP E3 ubiquitin ligases. These ligases ubiquitinate specifically phosphorylated substrates and play a pivotal role in the regulation of cell division and various signal transduction pathways, which, in turn, are essential for many aspects of tumorigenesis. We review the functions of the SCFbeta-TrCP ligases in the light of their relevance to cell growth, survival and transformation. Mechanisms underlying beta-TrCP regulation and their aberration in human and animal cancer as well as prospective of targeting beta-TrCP as a means of anticancer therapy are also discussed.

Animals↗
PubMed2005

Overexpression of human beta TrCP1 deleted of its F box induces tumorigenesis in transgenic mice.

Genetic alterations affecting beta-catenin, adenomatous polyposis coli or axin proteins are associated with the pathogenesis of numerous human tumors. All these mutations result in the synthesis of unphosphorylated beta-catenin that escapes recognition by the beta transducin repeat protein (beta TrCP1), the receptor of an ubiquitin. The stabilized beta-catenin translocates to the nucleus and activates the transcription of genes crucial for tumorigenesis. Recent evidence implicates mutations and overexpresssion of beta TrCP1 in human prostate and colon tumors, respectively, suggesting that deregulated beta TrCP1 may be involved in tumorigenesis. To explore this possibility further, we generated transgenic mice that specifically express a dominant-negative mutant of beta TrCP1 (Delta F beta TrCP1) or full-length beta TrCP1 in intestine, liver and kidney. We found that 46% (16/35) of the transgenic mice that overexpressed the transgenes developed either intestinal adenomas (10/35) or hepatic (4/35) or urothelial (2/35) tumors. Immunohistological analysis of the tumors revealed that upregulation of cyclin D1, glutamine synthetase and chemotaxin 2 was associated with nuclear accumulation of beta-catenin. These results show that the overexpression of Delta F beta TrCP1 or beta TrCP1 in vivo induce tumors through beta-catenin activation.

Adenoma↗
PubMed2006

Stabilization of prolactin receptor in breast cancer cells.

The role of the hormone prolactin (PRL) in the pathogenesis of breast cancer is mediated by its cognate receptor (PRLr). Ubiquitin-dependent degradation of the PRLr that negatively regulates PRL signaling is triggered by PRL-mediated phosphorylation of PRLr on Ser349 followed by the recruitment of the beta-transducin repeats-containing protein (beta-TrCP) ubiquitin-protein isopeptide ligase. We report here for the first time that interaction between PRLr and beta-TrCP is less efficient in human breast cancer cells than in non-tumorigenic human mammary epithelial cells. Furthermore, we demonstrate that both PRLr degradation and PRLr phosphorylation on Ser349 are impaired in breast tumor cells and tissues, an observation that directly correlates with enhanced expression of the PRLr in malignant breast epithelium. These findings represent a novel mechanism through which altered PRLr stability may directly influence the pathogenesis of breast cancer.

Breast↗
PubMed2003

Studies on G-protein alpha.betagamma heterotrimer formation reveal a putative S-prenyl-binding site in the alpha subunit.

The alpha and betagamma subunits of heterotrimeric G-proteins contain specific lipid modifications, which are required for their biological function. However, the relevance of these modifications to the interactions within the heterotrimeric G-protein is not fully understood. In order to explore the role of the S-prenyl moiety of the isoprenylated betagamma dimer of retinal transducin, betagamma(t), in the formation of the heterotrimeric complex with the corresponding N-acylated alpha subunit, alpha(t), we employed purified fully processed subunits, which are soluble in aqueous solutions without detergents. Pertussis-toxin-mediated [(32)P]ADP-ribosylation of alpha(t) is strongly stimulated (approximately 10-fold) in the presence of betagamma(t) and can thus serve as a measure for heterotrimer formation. Using this assay, preincubation of alpha(t) with S-prenyl analogues containing farnesyl or geranylgeranyl moieties was found to inhibit heterotrimer formation in a dose-dependent manner. The inhibition was competitive and reversible, as indicated by its reversal upon increase of the betagamma(t) dimer concentration or by removal of the S-prenyl analogue using gel filtration. The competitive nature of the inhibition is supported by the marked attenuation of the inhibition when the S-prenyl analogue was added to alpha(t) together with or after betagamma(t). The inhibition does not involve interaction with the alpha(t) acyl group, since an S-prenyl analogue inhibited the [(32)P]ADP-ribosylation of an unlipidated alpha(t) mutant. These data suggest the existence of a hitherto unrecognized S-prenyl-binding site in alpha(t), which is critical for its interaction with prenylated betagamma(t).

Acetylcysteine↗
PubMed2003

Are prenyl groups on proteins sticky fingers or greasy handles?

This Commentary discusses the work of Dietrich et al. in this issue of the Biochemical Journal, which sheds new light on the biological roles of protein-bound prenyl groups by providing evidence that the alpha-subunit of the heterotrimeric G-protein transducin has a binding site for the geranylgeranyl group of the gamma-subunit.

Animals↗
PubMed1986

Monoclonal antibodies to the guanine-nucleotide binding proteins of adenylate cyclase.

Monoclonal antibodies (Mabs) to the stimulatory (Ns) and inhibitory (Ni) guanine nucleotide regulatory proteins associated with adenylate cyclase have been developed. Two Mabs (2A3 and 5G12), which are of the IgG2b subclass, recognize the beta-subunits (beta) of Ns, Ni and transducin. Iodinated beta can be immunoprecipitated by either Mab coupled to Affi-Gel 10 and this can be decreased by prior incubation of the Mabs with excess unlabelled beta. The Mabs stabilize the activated state of Ns while decreasing the rate of deactivation of activated Ns in the presence of beta.

Adenylyl Cyclases↗
PubMed1989

Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments.

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5'-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1989

Schiff-base deprotonation is mandatory for light-dependent rhodopsin phosphorylation.

The absorption of light by rhodopsin leads to the formation of an activated intermediate (R*) capable of catalysing the exchange of GTP for GDP in a retinal guanine-nucleotide-binding regulatory protein (transducin). The ability of R* to function as a catalyst is terminated by the rhodopsin kinase. The 10 nonactive-site lysine residues of rhodopsin can be reductively dimethylated to form permethylated rhodopsin (PMRh). This derivative is phosphorylated to the same extent as rhodopsin after photolysis. The monomethylation of the active-site lysine residue of PMRh yields active-site-methylated rhodopsin (AMRh). It had previously been shown, by using AMRh, that the formation of R* and its spectroscopic signature metarhodopsin II requires the photochemically induced deprotonation of the active-site Schiff base [Longstaff, Calhoon & Rando (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4209-4213]. Here it is demonstrated that active-site Schiff-base deprotonation is also mandatory in the formation of the form of photolyzed rhodopsin that is susceptible to phosphorylation by rhodopsin kinase. In terms of the spectroscopically defined rhodopsin intermediates, this means that only metarhodopsin II and possibly metarhodopsin III are the actual substrates for rhodopsin kinase.

Animals↗
PubMed1992

Domain mapping of the retinal cyclic GMP phosphodiesterase gamma-subunit. Function of the domains encoded by the three exons of the gamma-subunit gene.

Retinal rod-outer-segment phosphodiesterase (PDE) is a heterotetramer consisting of two similar, but not identical, catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). Previously, we have reported that the site of PDE alpha/beta interaction with PDE gamma is located within residues 54-87 [Cunnick, Hurt, Oppert, Sakamoto & Takemoto (1990) Biochem. J. 271, 721-727]. The site for PDE gamma interaction with transducin alpha (T alpha) was found to encompass residues 24-45 of PDE gamma [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. In order to identify binding sites and other functional domains of PDE gamma, the three peptides which are encoded by the three exons of the PDE gamma gene were synthesized chemically. These exons encode for residues 1-49, 50-62 and 63-87 of bovine PDE gamma [Piriev, Purishko, Khramtsov & Lipkin (1990) Dokl. Akad. Nauk. SSSR 315, 229-230]. The peptide encompassing residues 63-87 was inhibitory in a PDE assay, whereas peptides 1-49 and 50-62 had no effect. However, both peptides 1-49 and 63-87 bound to PDE alpha/beta in a solid-phase binding assay. Only peptide 1-49 bound to T alpha.GTP[S] (GTP[S] is guanosine 5'-[gamma-thio]triphosphate). These data confirm that the inhibitory region of PDE gamma is encoded by exon 3 (residues 63-87), whereas a separate binding site for PDE alpha/beta and for T alpha.GTP[S] is encoded by exon 1 (residues 1-49). To study further the structure-function relationship of PDE gamma, this entire protein and two mutants were chemically synthesized. One mutant (-CT) lacked residues 78-87, whereas another replaced tyrosine-84 with glycine (TYR-84). Whereas the synthetic PDE gamma inhibited PDE alpha/beta catalytic activity, the -CT and TVR-84 mutants did not. All three synthetic proteins bound to both PDE alpha/beta and and T alpha.GTP[S]. These data confirm the presence of an alternative binding site on PDE gamma and demonstrate the importance of tyrosine-84 in PDE gamma inhibitory activity.

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

Large-scale production and purification of the human green cone pigment: characterization of late photo-intermediates.

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.

Animals↗
PubMed2001

Multiple pertussis toxin-sensitive G-proteins can couple receptors to GIRK channels in rat sympathetic neurons when expressed heterologously, but only native G(i)-proteins do so in situ.

Although many G-protein-coupled neurotransmitter receptors are potentially capable of modulating both voltage-dependent Ca(2+) channels (I(Ca)) and G-protein-gated K(+) channels (I(GIRK)), there is a substantial degree of selectivity in the coupling to one or other of these channels in neurons. Thus, in rat superior cervical ganglion (SCG) neurons, M(2) muscarinic acetylcholine receptors (mAChRs) selectively activate I(GIRK) whereas M(4) mAChRs selectively inhibit I(Ca). One source of selectivity might be that the two receptors couple preferentially to different G-proteins. Using antisense depletion methods, we found that M(2) mAChR-induced activation of I(GIRK) is mediated by G(i) whereas M(4) mAChR-induced inhibition of I(Ca) is mediated by G(oA). Experiments with the beta gamma-sequestering peptides alpha-transducin and beta ARK1(C-ter) indicate that, although both effects are mediated by G-protein beta gamma subunits, the endogenous subunits involved in I(GIRK) inhibition differ from those involved in I(Ca) inhibition. However, this pathway divergence does not result from any fundamental selectivity in receptor-G-protein-channel coupling because both I(GIRK) and I(Ca) modulation can be rescued by heterologously expressed G(i) or G(o) proteins after the endogenously coupled alpha-subunits have been inactivated with Pertussis toxin (PTX). We suggest instead that the divergence in the pathways activated by the endogenous mAChRs results from a differential topographical arrangement of receptor, G-protein and ion channel.

Animals↗
PubMed1999

Activation of the rod G-protein Gt by the thrombin receptor (PAR1) expressed in Sf9 cells.

Functional coupling of the human thrombin receptor PAR1 (protease-activated receptor 1) with the retinal rod G-protein transducin (Gt, a member of the Gi family) was studied in a reconstituted system of membranes from Sf9 cells expressing the thrombin receptor and purified Gt from bovine rod outer segments. TRAP6-agonist-activated PAR1 interacts productively with the distant G-protein. Agonist-dependent Gt activation was measured using a real-time fluorimetric GTP[S]-binding assay and membranes from Sf9 cells. To characterize nucleotide-exchange catalysis by PAR1, we analyzed dependence on nucleotides, temperature and pH. Activation was inhibited by low GDP concentrations (IC50 = 5.2 +/- 1.5 microM at 5 microM GTP[S]), indicating that receptor-Gt coupling, followed by instantaneous GDP release, is rate limiting under the conditions (25 degrees C). Arrhenius plots of the temperature dependence reflect an apparent Ea of 60 +/- 3.5 kJ.mol-1. Evaluation of the pH/rate profiles of Gt activation indicates that the activating conformation of the receptor is determined by protonation of a titratable group with an apparent pKa of 6.4. This supports the idea that the active state of agonist-bound PAR1 depends on forced protonation, indicating possible analogies to the scheme established for rhodopsin.

Animals↗