Adenosine diphosphate (ADP), an important platelet agonist, acts through 2 G-protein-coupled receptors (GPCRs), P2Y(1) and P2Y(12), which signal through Gq and Gi, respectively. There is increasing evidence for cross-talk between signaling pathways downstream of GPCRs and here we demonstrate cross-talk between these 2 ADP receptors in human platelets. We show that P2Y(12) contributes to platelet signaling by potentiating the P2Y(1)-induced calcium response. This potentiation is mediated by 2 mechanisms: inhibition of adenylate cyclase and activation of phosphatidylinositol 3 (PI 3)-kinase. Furthermore, the Src family kinase inhibitor PP1 selectively potentiates the contribution to the calcium response by P2Y(12), although inhibition of adenylate cyclase by P2Y(12) is unaffected. Using PP1 in combination with the inhibitor of PI 3-kinase LY294002, we show that Src negatively regulates the PI 3-kinase-mediated component of the P2Y(12) calcium response. Finally, we were able to show that Src kinase is activated through P2Y(1) but not P2Y(12). Taken together, we present evidence for a complex signaling interplay between P2Y(1) and P2Y(12), where P2Y(12) is able to positively regulate P2Y(1) action and P2Y(1) negatively regulates this action of P2Y(12). It is likely that this interplay between receptors plays an important role in maintaining the delicate balance between platelet activation and inhibition during normal hemostasis.

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The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic internalization as well as internalization in response to stimulation with phorbol esters and ligand (SDF-1␣). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1␣ and phorbol esters (PMA) promoted rapid internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 internalization in response to both PMA and SDF. Both PMA-and SDF-mediated CXCR4 internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1␣. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA-and SDF-1␣-mediated receptor internalization. The ability of coexpressed GRK2 and arrestins to promote internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor internalization.

Despite a widely accepted role of arrestins as "uncouplers" of G protein-coupled receptor (GPCR) signaling, few studies have demonstrated the ability of arrestins to affect second messenger generation by endogenously expressed receptors in intact cells. In this study we demonstrate arrestin specificity for endogenous GPCRs in primary cultures of human airway smooth muscle (HASM). Expression of arrestin-green fluorescent protein (ARR2-GFP or ARR3-GFP) chimeras in HASM significantly attenuated isoproterenol (␤ 2 -adrenergic receptor (␤ 2 AR)-mediated)-and 5-(N-ethylcarboxamido)adenosine (A2b adenosine receptor-mediated)-stimulated cAMP production, with fluorescent microscopy demonstrating agonist-promoted redistribution of cellular ARR2-GFP into a punctate formation. Conversely, prostaglandin E 2 (PGE 2 )-mediated cAMP production was unaffected by arrestin-GFP, and PGE 2 had little effect on arrestin-GFP distribution. The pharmacological profile of various selective EP receptor ligands suggested a predominantly EP2 receptor population in HASM. Further analysis in COS-1 cells revealed that ARR2-GFP expression increased agonist-promoted internalization of wild type ␤ 2 AR and EP4 receptors, whereas EP2 receptors remained resistant to internalization. However, expression of an arrestin whose binding to GPCRs is largely independent of receptor phosphorylation (ARR2(R169E)-GFP) enabled substantial agonist-promoted EP2 receptor internalization, increased ␤ 2 AR internalization to a greater extent than did ARR2-GFP, yet promoted EP4 receptor internalization to the same degree as did ARR2-GFP. Signaling via endogenous EP4 receptors in CHO-K1 cells was attenuated by ARR2-GFP expression, whereas ARR2(R169E)-GFP expression in HASM inhibited EP2 receptor-mediated cAMP production. These findings demonstrate differential effects of arrestins in altering endogenous GPCR signaling in a physiologically relevant cell type and reveal a variable dependence on receptor phosphorylation in dictating arrestin-receptor interaction.

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) traf®cking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 mM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quanti®cation of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin-and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319±418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 mM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-22green¯uorescent protein (arrestin-22GFP) or arrestin-32GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin-and dynamin-dependent process. Keywords: arrestin, dynamin, internalization, metabotropic glutamate receptor. Widely distributed throughout the CNS, metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that play a critical role in glutamate-mediated neurotransmission and synaptic plasticity events (for review, see Schoepp et al. 1999). On the basis of their amino acid sequence similarity, pharmacology and signal transduction mechanisms, mGluRs have been classi®ed into three groups, with the group I mGluRs, mGluR1 and mGluR5, being coupled to G q and phospholipase C. Five splice variants of mGluR1 have been described, all of them differing in the length of their C-terminal tail (Pin et al. 1992;Pin and Duvoisin 1995). Upon prolonged agonist addition, most GPCRs undergo decreased agonist responsiveness, a process known as desensitization. Recent studies have shown that desensitization of mGluR1a is G protein-coupled receptor kinase (GRK) dependent and that receptor phosphorylation by GRKs likely uncouples the GPCR from G-protein (Dale et al. 2000;Sallese et al. 2000). Following receptor G-protein uncoupling many GPCRs are removed from the cell surface by a process known as internalization (Krupnick and Benovic 1998). For some receptors, such as the b 2 -adrenoceptor, the binding of arrestin to receptor following GRK-dependent phosphorylation, serves to target the receptor to clathrin-coated pits for internalization (Goodman et al. 1996). Arrestins act as adaptors between the phosphorylated receptor and components of the endocytic machinery, such as AP-2 and clathrin, both of which are major components of clathrin-coated pits (Goodman et al. 1996...