An investigation into the structural determinants of cannabinoid receptor ligand efficacy.
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1. A number of side-chain analogues of delta8-THC were tested in GTPgammaS binding assay in rat cerebellar membranes. O-1125, a saturated side-chain compound stimulated GTPgammaS binding with an Emax of 165.0%, and an EC50 of 17.4 nM. 2. O-1236, O-1237 and O-1238, three-enyl derivatives containing a cis carbon-carbon double bond in the side-chain, stimulated GTPgammaS binding, acting as partial agonists with Emax values ranging from 51.3-87.5% and EC50 values between 4.4 and 29.7 nM. 3. The stimulatory effects of O-1125, O-1236, O-1237 and O-1238 on GTPgammaS binding were antagonized by the CB1 receptor antagonist SR 141716A. The K(B) values obtained ranged from 0.11-0.21 mM, suggesting an action at CB1 receptors. 4. Five-ynyl derivatives (O-584, O-806, O-823, O-1176 and O-1184), each containing a carbon-carbon triple bond in the side-chain, did not stimulate GTPgammaS binding and were tested as potential cannabinoid receptor antagonists. 5. Each -ynyl compound antagonized the stimulatory effects of four cannabinoid receptor agonists on GTPgammaS binding. The K(B) values obtained, all found to be in the nanomolar range, did not differ between agonists or from cerebellar binding affinity. 6. In conclusion, alterations of the side-chain of the classical cannabinoid structure may exert a large influence on affinity and efficacy at the CB1 receptor. 7. Furthermore, this study confirms the ability of the GTPgammaS binding assay to assess discrete differences in ligand efficacies which potentially may not be observed using alternative functional assays, thus providing a unique tool for the assessment of the molecular mechanisms underlying ligand efficacies. (+info)
Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells.
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Thyroid hormone [L-thyroxine (T4)] rapidly induced phosphorylation and nuclear translocation (activation) of mitogen-activated protein kinase (MAPK) in HeLa and CV-1 cells in the absence of cytokine or growth factor. A pertussis toxin-sensitive and guanosine 5'-O-(3-thiotriphosphate)-sensitive cell surface mechanism responsive to T4 and agarose-T4, suggesting a G protein-coupled receptor, was implicated. Cells depleted of MAPK or treated with MAPK pathway inhibitors showed reduced activation of MAPK and of the signal transducer and activator of transcription STAT1alpha by T4; they also showed reduced T4 potentiation of the antiviral action of interferon-gamma (IFN-gamma). T4 treatment caused tyrosine-phosphorylated MAPK-STAT1alpha nuclear complex formation and enhanced Ser-727 phosphorylation of STAT1alpha, in the presence or absence of IFN-gamma. STAT1alpha-deficient cells transfected with STAT1alpha containing an alanine-for-serine substitution at residue 727 (STAT1alphaA727) showed minimal T4-stimulated STAT1alpha activation. IFN-gamma induced the antiviral state in cells containing wild-type STAT1alpha (STAT1alphawt) or STAT1alphaA727; T4 potentiated IFN-gamma action in STAT1alphawt cells but not in STAT1alphaA727 cells. T4-directed STAT1alpha Ser-727 phosphorylation is MAPK mediated and results in potentiated STAT1alpha activation and enhanced IFN-gamma activity. (+info)
Measurement of agonist and antagonist ligand-binding parameters at the human parathyroid hormone type 1 receptor: evaluation of receptor states and modulation by guanine nucleotide.
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Determination of ligand-binding constants for parathyroid hormone (PTH) receptors has been hampered by a lack of suitable experimental systems and mechanistic models for data analysis. In this study, ligand binding to the cloned human PTH-1 receptor was measured using membrane-based radioligand-binding assays. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) (10 microM) reduced binding of agonist radioligands [125I]rPTH(1-34) and [125I]PTHrP(1-36) but only to a limited extent (by 29 +/- 5 and 42 +/- 3%, respectively). Radiolabeled agonist dissociation was described by three and two phases in the absence and presence of GTPgammaS, respectively. GTPgammaS treatment removed a pseudoirreversible binding phase. Inhibition of radiolabeled antagonist ([125I]bPTH(3-34)) binding was measured using a 90-min incubation, which allowed binding of ligands to closely approach the asymptotic maximum. Agonist/[125I]bPTH(3-34) displacement curves were fitted best by assuming two independent affinity states, both in the presence and absence of GTPgammaS. After a 3-h incubation, binding of PTH agonists in the presence of GTPgammaS was described by a single affinity state, indicating the presence of slow components in the binding reaction. Antagonist binding was described by a single affinity state and was not significantly affected by GTPgammaS. The data were used to evaluate potential receptor-binding models. Although other models could not be excluded, all of the observations could be explained by assuming two binding sites on the receptor that recognize two corresponding sites on agonist ligands. Using the model, it was possible to estimate receptor-ligand-binding constants and to propose a direct method for identifying ligands that interact with a putative antagonist binding region of the receptor. (+info)
Synthesis and characterization of potent and selective agonists of the neuronal cannabinoid receptor (CB1).
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Two subtypes of the cannabinoid receptor (CB1 and CB2) are expressed in mammalian tissues. Although selective antagonists are available for each of the subtypes, most of the available cannabinoid agonists bind to both CB1 and CB2 with similar affinities. We have synthesized two analogs of N-arachidonylethanolamine (AEA), arachidonylcyclopropylamide (ACPA) and arachidonyl-2-chloroethylamide (ACEA), that bind to the CB1 receptor with very high affinity (KI values of 2.2 +/- 0.4 nM and 1.4 +/- 0.3 nM, respectively) and to the CB2 receptor with low affinity (KI values of 0.7 +/- 0.01 microM and 3.1 +/- 1.0 microM, respectively). Both ACPA and ACEA have the characteristics of agonists at the CB1 receptor; both inhibit forskolin-induced accumulation of cAMP in Chinese hamster ovary cells expressing the human CB1 receptor, and both analogs increase the binding of [35S]GTPgammaS to cerebellar membranes and inhibit electrically evoked contractions of the mouse vas deferens. ACPA and ACEA produce hypothermia in mice, and this effect is inhibited by coadministration of the CB1 receptor antagonist SR141716A. Therefore, ACPA and ACEA are high-affinity agonists of the CB1 receptor but do not bind the CB2 receptor, suggesting that structural analogs of AEA can be designed with considerable selectivity for the CB1 receptor over the CB2 receptor. (+info)
Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration.
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Aberrant regulation of smooth muscle cell proliferation and migration is associated with the pathophysiology of vascular disorders such as hypertension, atherosclerosis, restenosis, and graft rejection. To elucidate molecular mechanisms that regulate proliferation and migration of vascular smooth muscle cells, we determined whether signaling through the small G protein Rho is involved in thrombin- and phenylephrine-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). Thrombin and the thrombin peptide SFLLRNP stimulated DNA synthesis of RASMCs as measured by [3H]thymidine incorporation. Both ligands also increased cell migration as measured by the Boyden chamber method. L-Phenylephrine failed to induce either of these responses but increased inositol phosphate accumulation and mitogen-activated protein kinase activation in these cells, which indicated that the cells were responsive to alpha1-adrenergic stimulation. The C3 exoenzyme, which ADP-ribosylates and inactivates Rho, fully inhibited both thrombin-stimulated proliferation and migration but had no effect on inositol phosphate accumulation. In addition, Y-27632, an inhibitor of the Rho effector p160ROCK/Rho kinase, decreased thrombin-stimulated DNA synthesis and migration. To directly examine Rho activation, Rho-[35S]GTPgammaS binding was measured. The addition of the thrombin peptide SFLLRNP, but not phenylephrine, to RASMC lysates resulted in a significant increase in Rho-[35S]GTPgammaS binding. Thrombin and SFLLRNP, but not phenylephrine, also increased membrane-associated Rho in intact RASMCs, consistent with selective activation of Rho by thrombin. These results indicate that thrombin activates Rho in RASMCs and establish Rho as a critical mediator of thrombin receptor effects on DNA synthesis and cell migration in these cells. (+info)
Peptides derived from the human transferrin receptor stimulate endosomal acidification via a Gi-type protein.
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Peptides derived from the human transferrin receptor stimulate endosomal acidification via a Gi-type protein. BACKGROUND: Acidification of the endosomal compartment is a prerequisite for intracellular processing of endocytosed complexes. Endosomal acidification is accomplished by an H+-ATPase, in parallel with a Cl- conductance. Previous studies from our laboratory have demonstrated that endosomal acidification is modulated by a pertussis toxin-sensitive mechanism, suggesting that endosomal acidification could be regulated through a self-contained signal transduction pathway. This study was designed to test this hypothesis using the transferrin receptor as a model. METHODS: Synthetic peptides corresponding to a region of the cytosolic domain of the transferrin receptor and containing a KPKR sequence were used to stimulate endosomal acidification in a G-protein-dependent manner. RESULTS: Peptides activated the Gi, as evidenced by stimulation of the rate of GTPgammaS binding. A transferrin receptor peptide that lacked the KPKR sequence did not stimulate endosomal acidification and failed to promote GTPgammaS binding to Gi proteins. CONCLUSIONS: These results demonstrate that regulation of endosomal acidification can be achieved, in part, through a Gi-mediated signal transduction pathway. These findings suggest that regulation of endosomal acidification through such a pathway may facilitate intracellular processing of the transferrin receptor. (+info)
Structure of Gialpha1.GppNHp, autoinhibition in a galpha protein-substrate complex.
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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)
Similar structures and shared switch mechanisms of the beta2-adrenoceptor and the parathyroid hormone receptor. Zn(II) bridges between helices III and VI block activation.
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The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A. , Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the beta2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works. (+info)