Vagus nerve modulates secretin binding sites in the rat forestomach.
(1/558)
Secretin is well known for its inhibitory action on gastric motility. It has been reported that secretin in a physiological dose inhibits gastric motility through mediation by the vagal afferent pathway. Secretin also elicited relaxation of carbachol-stimulated rat forestomach muscle strips by binding to its receptors, suggesting a direct action on this peripheral tissue. We hypothesized that vagal input may affect the action of secretin by modulating the level of secretin receptor in the forestomach. Several treatments, including vagal ligation, vagotomy, perivagal application of capsaicin or colchicine, intravenous infusion of tetrodotoxin, and intraperitoneal injection of atropine, were performed to investigate their effects on secretin receptor binding to forestomach membranes. Specific binding of 125I-labeled secretin to forestomach membranes was significantly decreased (45%) by vagal ligation, vagotomy (50%), or perivagal colchicine treatment (40%). On the contrary, specific binding of 125I-secretin was not affected by perivagal capsaicin treatment, intravenous infusion of tetrodotoxin, or intraperitoneal injection of atropine. By Scatchard analysis of the binding data, the capacity of the high-affinity binding sites in forestomach membranes was found to decrease significantly after vagal ligation compared with membranes from the sham-operated group. However, the affinity at the high-affinity binding sites, the binding parameters of the low-affinity binding sites, and binding specificity were not changed. Vagal ligation but not perivagal capsaicin treatment reduced the inhibitory effect of secretin on bethanechol-stimulated contraction of isolated forestomach muscle strips, causing a right shift in the dose-response curve. These results suggest that vagal input through axonal transport plays a significant role on secretin action by modulating the capacity of secretin binding sites (but not affinity or specificity), at least in rat forestomach. (+info)
Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal.
(2/558)
The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively. (+info)
A gain-of-function polymorphism in a G-protein coupling domain of the human beta1-adrenergic receptor.
(3/558)
The beta1-adrenergic receptor (beta1AR) is a key cell surface signaling protein expressed in the heart and other organs that mediates the actions of catecholamines of the sympathetic nervous system. A polymorphism in the intracellular cytoplasmic tail near the seventh transmembrane-spanning segment of the human beta1AR has been identified in a cohort of normal individuals. At amino acid position 389, Gly or Arg can be found (allele frequencies 0.26 and 0. 74, respectively), the former previously considered as the human wild-type beta1AR. Using site-directed mutagenesis to mimic the two variants, CHW-1102 cells were permanently transfected to express the Gly-389 and Arg-389 receptors. In functional studies with matched expression, the Arg-389 receptors had slightly higher basal levels of adenylyl cyclase activities (10.7 +/- 1.2 versus 6.1 +/- 0.4 pmol/min/mg). However, maximal isoproterenol-stimulated levels were markedly higher for the Arg-389 as compared to the Gly-389 receptor (63.3 +/- 6.1 versus 20.9 +/- 2.0 pmol/min/mg). Agonist-promoted [35S]guanosine 5'-O-(thiotriphosphate) binding was also increased with the Arg-389 receptor consistent with enhanced coupling to Gs and increased adenylyl cyclase activation. In agonist competition studies carried out in the absence of guanosine 5'-(beta, gamma-imido)triphosphate, high affinity binding could not be resolved with the Gly-389 receptor, whereas Arg-389 displayed an accumulation of the agonist high affinity receptor complex (RH = 26%). Taken together, these data indicate that this polymorphic variation of the human beta1AR results in alterations of receptor-Gs interaction with functional signal transduction consequences, consistent with its localization in a putative G-protein binding domain. The genetic variation of beta1AR at this locus may be the basis of interindividual differences in pathophysiologic characteristics or in the response to therapeutic betaAR agonists and antagonists in cardiovascular and other diseases. (+info)
Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.
(4/558)
The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here. (+info)
Structure of Gialpha1.GppNHp, autoinhibition in a galpha protein-substrate complex.
(5/558)
The structure of the G protein Gialpha1 complexed with the nonhydrolyzable GTP analog guanosine-5'-(betagamma-imino)triphosphate (GppNHp) has been determined at a resolution of 1.5 A. In the active site of Gialpha1. GppNHp, a water molecule is hydrogen bonded to the side chain of Glu43 and to an oxygen atom of the gamma-phosphate group. The side chain of the essential catalytic residue Gln204 assumes a conformation which is distinctly different from that observed in complexes with either guanosine 5'-O-3-thiotriphosphate or the transition state analog GDP.AlF4-. Hydrogen bonding and steric interactions position Gln204 such that it interacts with a presumptive nucleophilic water molecule, but cannot interact with the pentacoordinate transition state. Gln204 must be released from this auto-inhibited state to participate in catalysis. RGS proteins may accelerate the rate of GTP hydrolysis by G protein alpha subunits, in part, by inserting an amino acid side chain into the site occupied by Gln204, thereby destabilizing the auto-inhibited state of Galpha. (+info)
Effector recognition by the small GTP-binding proteins Ras and Ral.
(6/558)
The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar. (+info)
Sequestration of G-protein beta gamma subunits by different G-protein alpha subunits blocks voltage-dependent modulation of Ca2+ channels in rat sympathetic neurons.
(7/558)
The membrane-delimited and voltage-dependent inhibition of N-type Ca2+ channels is mediated by Gbeta gamma subunits. Previously, exogenous excess GDP-bound GalphaoA has been shown to dramatically attenuate the norepinephrine (NE)-mediated Ca2+ current inhibition by sequestration of Gbeta gamma subunits in rat superior cervical ganglion (SCG) neurons. In the present study, we determined whether the attenuation of NE-mediated modulation is specific to GalphaoA or shared by a number of closely related (Galphatr, GalphaoB, Galphai1, Galphai2, Galphai3, Galphaz) or unrelated (Galphas, Galphaq, Galpha11, Galpha16, Galpha12, Galpha13) Galpha subunits. Individual Galpha subunits from different subfamilies were transiently overexpressed in SCG neurons by intranuclear injection of mammalian expression vectors encoding the desired protein. Strikingly, all Galpha subunits except Galphaz nearly blocked basal facilitation and NE-mediated modulation. Likewise, VIP-mediated Ca2+ current inhibition, which is mediated by cholera toxin-sensitive G-protein, was also completely suppressed by a number of Galpha subunits overexpressed in neurons. Galphas expression produced either enhancement or attenuation of the VIP-mediated modulation-an effect that seemed to depend on the expression level. The onset of the nonhydrolyzable GTP analog, guanylylimidodiphosphate-mediated facilitation was significantly delayed by overexpression of different GDP-bound Galpha subunits. Taken together, these data suggest that a wide variety of Galpha subunits are capable of forming heterotrimers with endogenous Gbeta gamma subunits mediating voltage-dependent Ca2+ channel inhibition. In conclusion, coupling specificity in signal transduction is unlikely to arise as a result of restricted Galpha/Gbeta gamma interaction. (+info)
Structural view of the Ran-Importin beta interaction at 2.3 A resolution.
(8/558)
Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2. (+info)