Opiate tolerance and dependence are major clinical and social problems. The anti-opiate neuropeptides FF and AF (NPFF and NPAF) have been implicated in pain modulation as well as in opioid tolerance and may play a critical role in this process, although their mechanism of action has remained unknown. Here we describe a cDNA encoding a novel neuropeptide Y-like human orphan G protein-coupled receptor (GPCR), referred to as HLWAR77 for which NPAF and NPFF have high affinity. Cells transiently or stably expressing HLWAR77 bind and respond in a concentration-dependent manner to NPAF and NPFF and are also weakly activated by FMRF-amide (Phe-Met-Arg-Phe-amide) and a variety of related peptides. The high affinity and potency of human NPFF and human NPAF for HLWAR77 strongly suggest that these are the cognate ligands for this receptor. Expression of HLWAR77 was demonstrated in brain regions associated with opiate activity, consistent with the pain-modulating activity of these peptides, whereas the expression in adipose tissue suggests other physiological and pathophysiological activities for FMRF-amide neuropeptides. The discovery that the anti-opiate neuropeptides are the endogenous ligands for HL-WAR77 will aid in defining the physiological role(s) of these ligands and facilitate the identification of receptor agonists and antagonists.
b-Arrestin 1-GFP or b-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melaninconcentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both b-arrestin 1-GFP and b-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for b-arrestin 2-GFP. b-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of b-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and traf®cking of TAMRA-(5-and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of b-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of b-arrestin 1-GFP from intracellular vesicles. For the melaninconcentrating hormone receptor the bulk of translocated b-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of b-arrestin±GFP interactions and traf®cking for three G protein-coupled receptors for which the natural ligands have only recently been identi®ed and which were thus previously considered as orphan receptors.
A fusion protein was constructed between the porcine rx 2 A-adrenoceptor and a pertussis toxin-insensitive (Cys 351 Gly) form of the a subunit of the G protein GÜ . Addition of agonist ligands to membranes of COS-7 cells transiently transfected to express this construct, and treated with pertussis toxin prior to cell harvest, resulted in stimulation of both high affinity GTPase activity and enhanced binding of [ 35 S1GTPTS. By considering the fusion protein as an agonist-activated enzyme and measuring ^max of GTP hydrolysis a range of agonist ligands displayed varying efficacy in their capacity to activate the receptorassociated G protein with adrenaline = noradrenaline = rx-methylnoradrenaline > UK14304 > BHT933 > xylazine = clonidine. A similar rank order was observed following independent coexpression of the a 2 A-adrenoceptor and Cys 351 Gly-Giia. These data demonstrate the utility and applicability of using a receptor-G protein fusion protein approach, in which the stoichiometry of receptor and G protein is fixed at 1:1, to measure and further understand the nature of agonist efficacy.
Coexpression of the rat thyrotropin releasing hormone receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the receptor. Truncations of the receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the receptor. Despite this, the receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing hormone receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.
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