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G protein-coupled receptors (GPCRs) belong to a large family of cell-surface receptors involved in many intracellular signaling pathways. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (TKR) occurs in response to binding of specific agonists of several such receptors. Crosstalk between GPCRs and TKRs may occur by different molecular mechanism such as metalloproteases activation, non-receptor tyrosine kinases involvement, or reactive oxygen species (ROS) production, a process mediated by theNADPH oxidase enzymatic complex. Formyl-peptide receptors (FPRs) are seven transmembrane domain receptors, coupled to heterotrimeric Gi proteins and sensible to pertussis toxin (PTX). FPR1, FPR2 and FPR3 expression was observed in a wide variety of tissues and it has been implicated in tissue repair, angiogenesis and cancer. We investigated FPR1-mediated VEGFR2 transactivation and its involvement in angiogenesis processes in ECV304 endothelial cells. We demonstrated, by RT-PCR, that ECV304 cell line expresses p47phox, p67phox and p22phox subunits of NADPH oxidase, as well as FPR1, FPR3, VEGFR1 and VEGFR2 receptors. Stimulation of ECV304 with the FPR1 agonist N-fMLP induces p47phox phosphorylation, which is considered the key event of NADPH oxidase activation. Preincubation with PTX prevents p47phox phosphorylation. FPR1 stimulation by N-fMLP also triggers Y951, Y996 and Y1175 phosphorylation of cytosolic residues of VEGFR2, which elicits intracellular signaling cascades responsible of angiogenesis. Preincubation with PTX or FPR1 siRNA, or the inhibition of NADPH oxidase functions by apocynin or p22phox siRNA, prevents VEGFR2 tyrosine phosphorylation and, in turn, downstream signaling cascades.

The calcitonin gene related peptide (CGRP) is a 37 amino acid neuropeptide. Its receptor is a heterodimeric complex of calcitonin receptor-like receptor (CLR) – a family B G-protein coupled receptor – and a single-pass transmembrane protein, receptoractivity modifying protein 1 (RAMP1). Here, we identify residues, within the N-terminal extracellular domain (ECD) of CLR, potentially involved in ligand binding.Certain residues presumed to be possible sites of contact for the CGRP were picked from the CLR/RAMP1 ECD crystal structure (PDB 3N7S). Residues were mutated to alanine (A) bysite-directed mutagenesis (QuikChangeTM, Stratagene). Mutants were analysed for their ability to stimulate cAMP and cell surface expression as previously described [1]. All mutants showed reduced potency, though to varying degrees as indicated by their pEC50 values. W69A and D70Ashowed significant reduction in cell surface expression.These findings suggest that these residues are important for the interaction of CGRP with its receptor. W69A and D70A, part of the WDG motif of family B GPCRs, are thought to rather play a role in receptor stability [2]. The data is consistent with CGRP binding in agroove between CLR and RAMP1. This project was supported byAston School of Life and Health Sciences.References1. Barwell J, Conner A & Poyner D (2011) Extracellular loops 1and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim Biophys Acta 1813, 1906–1916.2. Kumar S, Pioszak A, Zhang C et al. (2011) Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Cou-pled Receptors. PLoS One 6, e19682

The effect of synthetic LVV-hemorphin-7 and hemorphin-7 on hypothalamo-pituitary-adrenocortical axis activity in response to endotoxin-induced stress was studied. The intraperitoneal (ip) endotoxin (lipopolysaccaride, LPS) (0.5 mg/kg) administration in combination with hemorphin (1 mg/kg) induce significant decrease in plasma corticosterone and modest decrease in plasma levels of tumor necrosis factor-alpha (TNFalpha) in compare with elevated levels of both corticosterone and TNFalpha in plasma of rats received LPS administration alone. Increased activity of calcineurin in both plasma and brain of rats received ip administration of LPS, was recovered under LPS + hemorphin treatment. In two independent proteome analysis, using 2-dimensional fluorescence difference gel electrophoresis and the isotope coded protein label technology, peptidyl-prolyl cis-trans-isomerase A (cyclophilin A) was identified as regulated by hemorphins protein in mouse brain. A therapeutic potential of hemorphins and mechanisms of their homeostatic action in response to endotoxin-induced stress are discussed.