During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G proteincoupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology. Keywords: G protein-coupled receptors, G protein-coupled receptor associated sorting protein, opioid receptors, proteinprotein interaction domain, yeast two-hybrid. G protein-coupled receptors (GPCRs) form one of the largest gene family in the human genome (see Pierce et al. 2002). The most recent estimation indicates that, excepting the odorant-receptor subfamily that contains several hundred members, the repertoire of GPCRs responding to endogenous ligands consists of 367 receptors in human and 392 in mouse (Vassilatis et al. 2003). These receptors are involved in the regulation of numerous physiological processes and represent major targets for pharmaceutical drugs. Upon ligand binding, GPCRs trigger numerous cellular events, including modification of second messenger levels (see Gudermann et al. 1997), receptor desensitization and internalization (see Pierce et al. 2002) and modification of gene transcription (see Pierce and Lefkowitz 2001;West et al. 2002). The molecular mechanisms underlying these phenomena are not completely understood and are the subject of intensive research.In the last few years, several novel proteins that physically interact with GPCRs have been identified, confirming that signal transduction associated with GPCRs is not restricted to heterotrimeric G protein activation (Hall et al. 1999;Brady and Limbird 2002). These associated proteins interact, either specifically with a small subset, or with a broad range of receptors, depending on the receptor region involved in the interaction. In most cases, this region is locate...
G protein-coupled receptor (GPCR) associated sorting protein 1 (GASP-1) interacts with GPCRs and is implicated in their postendocytic sorting. Recently, GASP-1 has been shown to regulate dopamine (D(2)) and cannabinoid (CB1) receptor signalling, suggesting that preventing GASP-1 interaction with GPCRs might provide a means to limit the decrease in receptor signalling upon sustained agonist treatment. In order to test this hypothesis, we have generated and behaviourally characterized GASP-1 knockout (KO) mice and have examined the consequences of the absence of GASP-1 on chronic cocaine treatments. GASP-1 KO and wild-type (WT) mice were tested for sensitization to the locomotor effects of cocaine. Additional mice were trained to acquire intravenous self-administration of cocaine on a fixed ratio 1 schedule of reinforcement, and the motivational value of cocaine was then assessed using a progressive ratio schedule of reinforcement. The dopamine and muscarinic receptor densities were quantitatively evaluated in the striatum of WT and KO mice tested for sensitization and self-administration. Acute and sensitized cocaine-locomotor effects were attenuated in KO mice. A decrease in the percentage of animals that acquired cocaine self-administration was also observed in GASP-1-deficient mice, which was associated with pronounced down-regulation of dopamine and muscarinic receptors in the striatum. These data indicate that GASP-1 participates in acute and chronic behavioural responses induced by cocaine and are in agreement with a role of GASP-1 in postendocytic sorting of GPCRs. However, in contrast to previous studies, our data suggest that upon sustained receptor stimulation GASP-1 stimulates recycling rather than receptor degradation.
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