Identification of residues important for agonist recognition and activation in GPR40

GPR40 was formerly an orphan G protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids (FFAs). The receptor, now named FFA receptor 1, has been implicated in the pathophysiology of type 2 diabetes and is a drug target because of its role in FFA-mediated enhancement of glucose-stimulated insulin release. Guided by molecular modeling, we investigated the molecular determinants contributing to binding of linoleic acid, a C18 polyunsaturated FFA, and GW9508, a synthetic small molecule agonist. Twelve residues within the putative GPR40-binding pocket including hydrophilic/positively charged, aromatic, and hydrophobic residues were identified and were subjected to site-directed mutagenesis. Our results suggest that linoleic acid and GW9508 are anchored on their carboxylate groups by Arg183, Asn244, and Arg258. Moreover, His86, Tyr91, and His137 may contribute to aromatic and/or hydrophobic interactions with GW9508 that are not present, or relatively weak, with linoleic acid. The anchor residues, as well as the residues Tyr12, Tyr91, His137, and Leu186, appear to be important for receptor activation also. Interestingly, His137 and particularly His86 may interact with GW9508 in a manner dependent on its protonation status. The greater number of putative interactions between GPR40 and GW9508 compared with linoleic acid may explain the higher potency of GW9508.

Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin

The cholecystokinin (CCK) receptor-2 exerts very important central and peripheral functions by binding the neuropeptides cholecystokinin or gastrin. Because this receptor is a potential therapeutic target, great interest has been devoted to the identification of efficient antagonists. However, interspecies genetic polymorphism that does not alter cholecystokinin-induced signaling was shown to markedly affect activity of synthetic ligands. In this context, precise structural study of the agonist binding site on the human cholecystokinin receptor-2 is a prerequisite to elucidating the molecular basis for its activation and to optimizing properties of synthetic ligands. In this study, using site-directed mutagenesis and molecular modeling, we delineated the binding site for CCK on the human cholecystokinin receptor-2 by mutating amino acids corresponding to that of the rat homolog. By doing so, we demonstrated that, although resembling that of rat homolog, the human cholecystokinin receptor-2 binding site also displays important distinct structural features that were demonstrated by susceptibility to several point mutations (F120A, Y189A, H207A). Furthermore, docking of CCK in the human and rat cholecystokinin receptor-2, followed by dynamic simulations, allowed us to propose a plausible structural explanation of the experimentally observed difference between rat and human cholecystokinin-2 receptors.

Insulin-releasing and metabolic effects of small molecule GLP-1 receptor agonist 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline

Much recent attention has focused on the GLP-1 receptor as a potential target for antidiabetic drugs. Enzyme resistant GLP-1 mimetics such as exenatide are now employed for the treatment of type 2 diabetes, but must be administered by injection. The present study has examined and compared the in vitro and in vivo metabolic actions of a small molecule GLP-1 receptor agonist 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB), with native GLP-1, exenatide and liraglutide. DMB significantly stimulated in vitro insulin secretion from BRIN-BD11 cells but with decreased molar potency compared to native GLP-1 or related mimetics. Administration of DMB in combination with glucose to mice significantly (P

Platelet 12-lipoxygenase activation via glycoprotein VI - Involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H( P) ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 mug/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI ( GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H( P) ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H( P) ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P) ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists), This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples, The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELISA results. Four calves were orally treated with RCT (0.1 mg kg(-1) body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli), High concentrations of RCT residues were found throughout the medication period (44-473 ng ml(-1); LC-MS-MS data) and remained present for several days following removal of the drug from the diet, RCT residues were no longer detectable 2 weeks after withdrawal, Good agreement (r(2) = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for both sets of analyses, The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal, However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.

EFFECTIVE LABORATORY MONITORING FOR THE ABUSE OF THE BETA-AGONIST CLENBUTEROL IN CATTLE

The use of the beta-agonist clenbuterol (CBL) as a growth promoter has been outlawed in European meat production. The detection of its illegal use is dependent on CBL residues persisting in animal tissues for longer than the withdrawal times given by abusers. A comparison of urine, bile and liver matrices indicated that analysis of the liver offered the best possibility for CBL detection. However, an experimental study showed that CBL detection following withdrawal could be further extended (up to 56 d) if the retina was used as the target tissue. Analysis of 703 retina and liver samples from cattle suspected of CBL medication revealed that 96 cattle had CBL residues present in their retinas, only 46 of these were liver positive. There were no instances of liver CBL residues being detected without the associated retina also being positive.