Molecular pharmacology
-
Molecular pharmacology · Aug 2004
Interleukin-1beta-induced mucin production in human airway epithelium is mediated by cyclooxygenase-2, prostaglandin E2 receptors, and cyclic AMP-protein kinase A signaling.
We reported recently that interleukin (IL)-1beta exposure resulted in a prolonged increase in MUC5AC mucin production in normal, well differentiated, human tracheobronchial epithelial (NHTBE) cell cultures, without significantly increasing MUC5AC mRNA (Am J Physiol 286:L320-L330, 2004). The goal of the present study was to elucidate the signaling pathways involved in IL-1beta-induced MUC5AC production. We found that IL-1beta increased cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin (PG) E(2) production and that the COX-2 inhibitor celecoxib suppressed IL-1beta-induced MUC5AC production. ⋯ However, neither inhibition of epidermal growth factor receptor (EGFR) activation with the tyrosine kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline HCl (AG-1478) or EGFR blocking antibody nor inhibition of extracellular signal-regulated kinase/P-38 mitogen activated protein kinases with specific inhibitors blocked IL-1beta stimulation of MUC5AC mucin production. We also observed that tumor necrosis factor (TNF)-alpha, platelet activating factor (PAF), and lipopolysaccharide (LPS) induced COX-2 and increased MUC5AC production that was blocked by celecoxib, suggesting a common signaling pathway of inflammatory mediator-induced MUC5AC production in NHTBE cells. We conclude that the induction of MUC5AC by IL-1beta, TNF-alpha, PAF, and LPS involves COX-2- generated PGE(2), activation of EP2 and/or EP4 receptor(s), and cAMP-PKA-mediated signaling.
-
Molecular pharmacology · Aug 2004
Identification of a novel site within G protein alpha subunits important for specificity of receptor-G protein interaction.
Several domains of G protein alpha subunits are implicated in the control of receptor-G protein coupling specificity. Among these are the extreme N-and C-termini, the alpha4/beta6-loops, and the loop linking the N-terminal alpha-helix to the beta1-strand of the ras-like domain. In this study, we illustrate that single-point mutations of a highly conserved glycine residue within the linker I region of the Galpha(q) subunit confers upon the mutant Galpha(q) the ability to be activated by Galpha(i)- and Galpha(s) -coupled receptors, as evidenced by guanosine 5'-O-(3-[(35)S]thio)triphosphate binding and inositol phosphate turnover assays. ⋯ It is noteworthy that both mutant and wild-type Galpha(q) proteins are indistinguishable in their ability to reconstitute a functional Gq-PLCbeta-calcium signaling pathway when cotransfected with the Galpha(q)-coupled neurokinin 1 or muscarinic M3 receptor into mouse embryonic fibroblasts derived from Galpha(q/11) knockout mice. On a three-dimensional model of the receptor-G protein complex, the highly conserved linker I region connecting the helical and the GTPase domain of the Galpha protein is inaccessible to the intracellular surface of the receptors. Our data indicate that receptor-G protein coupling specificity is not exclusively governed by direct receptor-G protein interaction and that it even bypasses the requirement of the extreme C terminus of Galpha, a well accepted receptor recognition domain, suggesting a novel allosteric mechanism for G protein-coupled receptor-G protein selectivity.
-
Molecular pharmacology · Jul 2004
The structural basis for GTS-21 selectivity between human and rat nicotinic alpha7 receptors.
The alpha7 nAChR-selective partial agonist 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is more efficacious and potent for rat receptors than for human alpha7 receptors. Four single amino acid differences exist between human and rat alpha7 in the agonist binding site, two in the C loop, and one each in the E and F loops. Reciprocal mutations were made in these three domains and evaluated in Xenopus laevis oocytes. ⋯ However, the reversal of the potency ratio seen with the EF mutants was not evident in the CEF mutants. Our data therefore indicate that the pharmacological differences between rat and human alpha7 receptors are caused by reciprocal differences in sites within and around the binding site. Specific features in the agonist molecule itself are also identified that interact with these structural features of the receptor agonist binding site.
-
Molecular pharmacology · May 2004
Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).
We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. ⋯ Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.
-
Molecular pharmacology · Mar 2004
Vasopressin stimulates insulin release from islet cells through V1b receptors: a combined pharmacological/knockout approach.
Vasopressin receptor subtype(s) responsible for stimulation of insulin release from pancreatic beta cells were investigated by using subtype-selective antagonists and mice that were genetically lacking either V1a or V1b receptors. Arginine vasopressin (AVP) increased insulin release from isolated mouse islet cells in a concentration-dependent manner, with a submaximal response at 100 nM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis detected V1b and oxytocin, but not V1a or V2, receptor transcripts in mouse islet cells. ⋯ The inhibitory effects of vasopressin antagonists on AVP-induced insulin release correlate well with the rank order of potency to inhibit [3H]AVP binding to the V1b receptor; pancreatic islet cells were significantly inhibited by SSR149415 but not by SR49059 or OPC-21268. Furthermore, the AVP effect on insulin release was entirely lost in mice lacking the V1b receptor but was preserved in mice lacking the V1a receptor. Our study, which combined pharmacological and knockout approaches, clearly demonstrates that vasopressin-stimulated insulin release from islet cells is mediated via V1b receptors.