Summary The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII and clathrin coats. Here we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. Biochemically, the BBSome is the major effector of the Arf-like GTPase Arl6/ BBS3. In vivo, the BBSome and Arl6 localize to ciliary punctae and Arl6GTP is required to target the BBSome to cilia. Congruently, GTP-bound Arl6 and acidic phospholipids are sufficient to efficiently recruit the BBSome to chemically defined liposomes. Finally, ultrastructural analyses demonstrate that BBSome binding to liposomes produces distinct patches of polymerized coat. Since we establish that the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome to mediate targeting to cilia, we propose that trafficking to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals.

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The chemokine Cxcl12 binds Cxcr4 and Cxcr7 receptors to control cell migration in multiple biological contexts, including brain development, leukocyte trafficking, and tumorigenesis. Both receptors are expressed in the CNS, but how they cooperate during migration has not been elucidated. Here, we used the migration of cortical interneurons as a model to study this process. We found that Cxcr4 and Cxcr7 are coexpressed in migrating interneurons, and that Cxcr7 is essential for chemokine signaling. Intriguingly, this process does not exclusively involve Cxcr7, but most critically the modulation of Cxcr4 function. Thus, Cxcr7 is necessary to regulate Cxcr4 protein levels, thereby adapting chemokine responsiveness in migrating cells. This demonstrates that a chemokine receptor modulates the function of another chemokine receptor by controlling the amount of protein that is made available for signaling at the cell surface.

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Opioid analgesics are powerful pain relievers; however, over time, pain control diminishes as analgesic tolerance develops. The molecular mechanisms initiating tolerance have remained unresolved to date. We have previously shown that desensitization of the μ-opioid receptor and interaction with β-arrestins is controlled by carboxyl-terminal phosphorylation. Here we created knockin mice with a series of serine- and threonine-to-alanine mutations that render the receptor increasingly unable to recruit β-arrestins. Desensitization is inhibited in locus coeruleus neurons of mutant mice. Opioid-induced analgesia is strongly enhanced and analgesic tolerance is greatly diminished. Surprisingly, respiratory depression, constipation, and opioid withdrawal signs are unchanged or exacerbated, indicating that β-arrestin recruitment does not contribute to the severity of opioid side effects and, hence, predicting that G-protein-biased µ-agonists are still likely to elicit severe adverse effects. In conclusion, our findings identify carboxyl-terminal multisite phosphorylation as key step that drives acute μ-opioid receptor desensitization and long-term tolerance.

Morphine is a poor inducer of l-opioid receptor (MOR) internalization, but a potent inducer of cellular tolerance. Here we show that, in contrast to full agonists such as [D-Ala 2 -MePhe 4 -Gly-ol]enkephalin (DAMGO), morphine stimulated a selective phosphorylation of the carboxyterminal residue 375 (Ser 375 ). Ser 375 phosphorylation was sufficient and required for morphine-induced desensitization of MOR. In the presence of full agonists, morphine revealed partial agonistic properties and potently inhibited MOR phosphorylation and internalization. Upon removal of the drug, DAMGO-desensitized receptors were rapidly dephosphorylated. In contrast, morphine-desensitized receptors remained at the plasma membrane in a Ser 375 -phosphorylated state for prolonged periods. Thus, morphine promotes terminal MOR desensitization by inducing a persistent modification of Ser 375 .

Differences in the ability of opioid drugs to promote regulated endocytosis of m-opioid receptors are related to their tendency to produce drug tolerance and dependence. Here we show that drugspecific differences in receptor internalization are determined by a conserved, 10-residue sequence in the receptor's carboxylterminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at serine (S)375, present in the middle of this sequence, but opioids differ markedly in their ability to drive higher-order phosphorylation on flanking residues [threonine (T)370, T376, and T379]. Multi-phosphorylation is required for the endocytosispromoting activity of this sequence and occurs both sequentially and hierarchically, with S375 representing the initiating site. Higherorder phosphorylation involving T370, T376, and T379 specifically requires GRK2/3 isoforms, and the same sequence controls opioid receptor internalization in neurons. These results reveal a biochemical mechanism differentiating the endocytic activity of opioid drugs.

Recent biochemical, biophysical, and functional studies suggest that G protein-coupled receptors (GPCRs) 1 can assemble as homo-or heterodimeric complexes (1, 2). Heterodimerization has been shown to alter both ligand binding affinity and signaling efficacy of GPCRs (1, 2). ␦-and -opioid receptors form stable heterodimers with ligand binding and signaling properties resembling that of the 2 receptor (3). Formation of heterodimers between the sst 1 and sst 5 somatostatin receptors has been found to modulate the pharmacology and signaling of both receptors (4). The ␥-aminobutyric acid receptor B is unique in that heterodimerization of the nonfunctional ␥-aminobutyric acid receptors B1 and B2 is required for native affinity for ligands and complete functional activity (5-9). Heteromeric assembly of fully functional AT 1 angiotensin II and B 2 bradykinin receptors results in increased efficacy of angiotensin II and decreased efficacy of bradykinin (10). Heterodimerization has also been shown to alter endocytotic trafficking of GPCRs (3, 4, 10, 11). The -␦ heterodimer exhibited a decrease in agonist-mediated receptor endocytosis (3). Oligomerization of ␦-and -opioid receptors with the distantly related ␤ 2 -adrenergic receptor results in increased and decreased receptor endocytosis, respectively (11). AT 1 -B 2 heterodimerization induced a switch to a clathrin-and dynamindependent endocytotic pathway for both receptors (10). Signaling of GPCRs is often terminated by phosphorylation of intracellular serine and threonine residues. After phosphorylation of the receptor, arrestins are frequently recruited to the plasma membrane, at which they facilitate endocytosis by serving as scaffolding proteins that bind to clathrin. Although changes in trafficking have been clearly documented, agonistinduced phosphorylation and desensitization of these GPCR heterodimers has not been examined.We have recently shown that the sst 2A and sst 3 somatostatin receptors exist as constitutive homodimers when expressed alone and as constitutive heterodimers when coexpressed in human embryonic kidney (HEK) 293 cells (12). Whereas the sst 2A -sst 3 heterodimer behaved like the sst 2A homodimer, it did not reproduce the pharmacological characteristics of the sst 3 homodimer, suggesting that physical interaction of sst 3 with sst 2A induced functional inactivation of the sst 3 subtype (12). Here we report that the sst 2A receptor also forms stable heterodimers with the -opioid receptor (MOR1), a member of a closely related GPCR family. Unlike that observed for the sst 2A -sst 3 heterodimer, sst 2A -MOR1 heterodimerization did not significantly affect the ligand binding or coupling properties but promoted cross-modulation of phosphorylation, internalization, and desensitization of these receptors. EXPERIMENTAL PROCEDURES Materials