The chemoattractant stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) are key modulators of immune function. In the developing brain, SDF-1 is crucial for neuronal guidance; however, cerebral functions of SDF-1/CXCR4 in adulthood are unclear. Here, we examine the cellular expression of SDF-1 isoforms and CXCR4 in the brain of mice receiving systemic lipopolysaccharide (LPS) or permanent focal cerebral ischemia. CXCR4 mRNA was constitutively expressed in cortical and hippocampal neurons and ependymal cells. Hippocampal neurons targeted the CXCR4 receptor to their somatodendritic and axonal compartments. In cortex and hippocampus, CXCR4-expressing neurons exhibited an overlapping distribution with neurons expressing SDF-1 transcripts. Although neurons synthesized SDF-1alpha mRNA, the SDF-1beta isoform was selectively expressed by endothelial cells of cerebral microvessels. LPS stimulation dramatically decreased endothelial SDF-1beta mRNA expression throughout the forebrain but did not affect neuronal SDF-1alpha. After focal cerebral ischemia, SDF-1beta expression was selectively increased in endothelial cells of penumbral blood vessels and decreased in endothelial cells of nonlesioned brain areas. In the penumbra, SDF-1beta upregulation was associated with a concomitant infiltration of CXCR4-expressing peripheral blood cells, including macrophages. Neuronal SDF-1alpha was transiently downregulated and neuronal CXCR4 was transiently upregulated in the nonlesioned cerebral cortex in response to ischemia. Although endothelial SDF-1beta may control cerebral infiltration of CXCR4-carrying leukocytes during cerebral ischemia, the neuronal SDF-1alpha/CXCR4 system may contribute to ischemia-induced neuronal plasticity. Thus, the isoform-specific regulation of SDF-1 expression modulates neurotransmission and cerebral infiltration via distinct CXCR4-dependent pathways.

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 .

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

The main analgesic effects of the opioid alkaloid morphine are mediated by the -opioid receptor. In contrast to endogenous opioid peptides, morphine activates the -opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse -opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [D-Ala 2 ,Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four -receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell-and tissuespecific C-terminal splicing of the -opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.Opioid receptors couple via G proteins to a variety of downstream effectors including adenylate cyclase (1) and mitogenactivated protein kinases (2-5). During repeated or continuous agonist stimulation these responses undergo rapid desensitization. An important mechanism of desensitization of G proteincoupled receptors is the phosphorylation of intracellular receptor domains by G protein-coupled receptor kinases or second messenger-regulated protein kinases such as Ca 2ϩ /calmodulindependent protein kinase II, cAMP-dependent protein kinase, or protein kinase C. 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 (6, 7). For some time it has been assumed that the rapid removal of ligand-activated receptors from the cell surface plays a major role in the receptor degradation, thus enhancing functional desensitization. Recent studies have suggested that endocytozed receptors are predominantly recycled to the cell surface in a reactivated state (8, 9).We have shown previously that two C-terminal splice variants (rMOR1 and rMOR1B) 1 of the rat -opioid receptor differ in their DAMGO-mediated internalization and resensitization rates (10, 11). The rapid internalizing variant rMOR1B revealed a faster resensitization and consequently a slower desensitization as compared with rMOR1. These results clearly show that receptor recycling after interna...

The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst 1 -sst 5 ) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of ␤-arrestin mobilization and endosomal sorting. The ␤-arrestin-dependent trafficking of the sst 2A somatostatin receptor resembled that of a class B receptor in that upon receptor activation, ␤-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst 2A receptor. Unlike other class B receptors, however, the sst 2A receptor was rapidly resensitized and recycled to the plasma membrane. The ␤-arrestin mobilization of the sst 3 and the sst 5 somatostatin receptors resembled that of a class A receptor in that upon receptor activation, ␤-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, ␤-arrestin was excluded from sst 3 -containing vesicles. Unlike other class A receptors, a large proportion of sst 3 receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst 4 somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and ␤-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst 2A and sst 3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst 2A Although G protein-coupled receptors (GPCRs) 1 generally were believed to act as monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. The existence of homodimers has been shown for several GPCRs including the ␤ 2 -adrenergic receptor (1), ␦-and -opioid receptors (2, 3), the metabotrobic glutamate receptor 5 (4), the calcium-sensing receptor (5), the m3 muscarinic receptor (6), and dopamine receptors (7). GPCRs seem to dimerize via different mechanisms. Whereas dimerization of the ␤ 2 -adrenergic receptor (1) and D2 dopamine receptor (8) occurs via their transmembrane regions, dimerization of the ␦-opioid receptor involves the carboxyl terminus (2). In contrast, the metabotrobic glutamate receptor 5 (4) and the calcium-sensing receptor (5, 9, 10) appear to be disulfide-linked dimers, and dimerization occurs via their large amino termini. The question of to what extent agonist binding affects dimerization remains controversial. Recent evidence obtained in living cells using bioluminescence resonance energy transfer suggests that the ␤ 2 -adrenergic receptor exists at the cell surface as a constitutive dimer that is stabilized by agonist binding (11). In contrast, agonist stimulation reduced the level of ␦-opioid receptor dimers suggesting that monomerization precedes agonist-induced internalization of this receptor (2). Biochemical and functional studies suggest that GPCRs can also assemble as heterodimers with enhanced functional activity (3,(12)(13)(14)(15)(16)(17)(18)(19)(20). Formation of heterodimers between two nonfunctional ␥-aminobutyric acid (GABA) receptors, GABA B R1 and GABA B R2, was necessary for a fully functional GABA B receptor (14 -19). ␦-and -Opioid receptors form heterodimers with ligand binding and signaling properties resembling that of the 2 receptor (3). Finally, the somatostatin receptor (sst) sst 5 and the D 2 dopamine receptor heterodimerize to form a new receptor with en-

The -opioid receptor (MOR1) and the substance P receptor (NK1) coexist and functionally interact in nociceptive brain regions; however, a molecular basis for this interaction has not been established. (14). Given the high level of sequence homology between opioid and somatostatin receptors existing in domains proposed to contribute to the dimerization interface (i.e. transmembrane helices) it was not unexpected that these closely related receptors can form heterodimers. However, so far little is known about dimerization of opioid receptors with distantly related GPCRs. A major prerequisite for the physiological assembly of heterodimeric GPCRs in vivo is their coexpression in the same cell. MOR1 and NK1, the principal receptor for substance P (SP), coexist and functionally interact in pain-processing brain regions (15)(16)(17)(18)(19)(20)(21)(22). SP is released from nociceptive primary afferents in the spinal and trigeminal dorsal horn, where it activates spino-thalamic projection neurons. Recently, NK1 and MOR1 have been shown to coexist in trigeminal dorsal horn neurons, suggesting that MOR and NK1 functionally interact in these neurons during nociceptive neurotransmission. Furthermore, MOR1 and NK1 are highly expressed in brain regions implicated in depression, anxiety, and stress, but also in other regions such as the nucleus accumbens, which mediate the motivational properties of drugs of abuse including opioids. Interestingly, the rewarding effects of morphine are absent in mice lacking the NK1 receptor (20,21). Despite these observations, a molecular basis for the interactions between opioid and substance P receptors has not been established.Here, we report that the MOR1 receptor forms stable heterodimers with the NK1 receptor. MOR1-NK1 heterodimerization did not substantially change ligand binding and signaling properties of the MOR1 receptor, but it dramatically altered its trafficking and resensitization profile.

Stromal-cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) play a well-established role during embryonic development of dentate gyrus granule cells. However, little is known about the regulation and function of CXCR4 in the postnatal dentate gyrus. Here, we identify a striking mismatch between intense CXCR4 mRNA and limited CXCR4 protein expression in adult rat subgranular layer (SGL) neurons. We demonstrate that CXCR4 protein expression in SGL neurons is progressively lost during postnatal day 15 (P15) to P21. This loss of CXCR4 protein expression was paralleled by a reduction in the number of SDF-1-responsive SGL neurons and a massive upregulation of SDF-1 mRNA in granule cells. Intraventricular infusion of the CXCR4-antagonist AMD3100 dramatically increased CXCR4 protein expression in SGL neurons, suggesting that CXCR4 is tonically activated and downregulated by endogenous SDF-1. Infusion of AMD3100 also facilitated detection of CXCR4 protein in bromodeoxyuridine-, nestin-, and doublecortin-labeled cells and showed that the vast majority of adult-born granule cells transiently expressed CXCR4. Chronic AMD3100 administration impaired formation of new granule cells as well as neurogenesis-dependent long-term recognition of novel objects. Therefore, our findings suggest that tonic activation of CXCR4 in newly formed granule cells by endogenous SDF-1 is essential for neurogenesis-dependent long-term memory in the adult hippocampus.