Differentiation of opioid drug effects by hierarchical multi-site phosphorylation.

Differences in the ability of opioid drugs to promote regulated endocytosis of µ-opioid receptors are related to their tendency to produce drug tolerance and dependence. Here we show that drug-specific differences in receptor internalization are determined by a conserved, 10-residue sequence in the receptor's carboxyl-terminal 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 endocytosis-promoting activity of this sequence and occurs both sequentially and hierarchically, with S375 representing the initiating site. Higher-order 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.

Desensitization and trafficking of µ-opioid receptors in locus ceruleus neurons: modulation by kinases.

The phosphorylation of µ-opioid receptors (MOPRs) by G protein-coupled receptor kinases (GRKs), followed by arrestin binding, is thought to be a key pathway leading to desensitization and internalization. The present study used the combination of intracellular and whole-cell recordings from rats and mice, as well as live cell imaging of Flag-tagged MOPRs from mouse locus ceruleus neurons, to examine the role of protein kinases in acute desensitization and receptor trafficking. Inhibition of GRKs by using heparin or GRK2-mutant mice did not block desensitization or alter the rate of recovery from desensitization. The nonselective kinase inhibitor staurosporine did not reduce the extent of [Met(5)]enkephalin (ME)-induced desensitization but increased the rate of recovery from desensitization. In the presence of staurosporine, ME-activated FlagMOPRs were internalized but did not traffic away from the plasma membrane. The increased rate of recovery from desensitization correlated with the enhancement in the recycling of receptors to the plasma membrane. ME-induced MOPR desensitization persisted and the trafficking of receptors was modified after inhibition of protein kinases. The results suggest that desensitization of MOPRs may be an early step after agonist binding that is modulated by but is not dependent on kinase activity.

Quantitative encoding of the effect of a partial agonist on individual opioid receptors by multisite phosphorylation and threshold detection.

In comparison to endogenous ligands of seven-transmembrane receptors, which typically act as full agonists, many drugs act as partial agonists. Partial agonism is best described as a "macroscopic" property that is manifest at the level of physiological systems or cell populations; however, whether partial agonists also encode discrete regulatory information at the "microscopic" level of individual receptors is not known. Here, we addressed this question by focusing on morphine, a partial agonist drug for µ-type opioid peptide receptors (MORs), and by combining quantitative mass spectrometry with cell biological analysis to investigate the reduced efficacy of morphine, compared to that of a peptide full agonist, in promoting receptor endocytosis. We showed that these chemically distinct ligands produced a complex and qualitatively similar mixture of phosphorylated opioid receptor forms in intact cells. Quantitatively, however, the different agonists promoted disproportionate multisite phosphorylation of a specific serine and threonine motif, and we found that modification at more than one residue was essential for the efficient recruitment of the adaptor protein ß-arrestin that mediated subsequent endocytosis of MORs. Thus, quantitative encoding of agonist-selective endocytosis at the level of individual opioid receptors was based on the conserved biochemical principles of multisite phosphorylation and threshold detection.

Recovery from mu-opioid receptor desensitization after chronic treatment with morphine and methadone.

Chronic treatment with morphine results in a decrease in µ-opioid receptor sensitivity, an increase in acute desensitization, and a reduction in the recovery from acute desensitization in locus ceruleus neurons. With acute administration, morphine is unlike many other opioid agonists in that it does not mediate robust acute desensitization or induce receptor trafficking. This study compares µ-opioid receptor desensitization and trafficking in brain slices taken from rats treated for 6-7 d with a range of doses of morphine (60, 30, and 15 mg · kg(-1) · d(-1)) and methadone (60, 30, and 5 mg · kg(-1) · d(-1)) applied by subcutaneous implantation of osmotic minipumps. Mice were treated with 45 mg · kg(-1) · d(-1). In morphine-treated animals, recovery from acute [Met](5)enkephalin-induced desensitization and receptor recycling was diminished. In contrast, recovery and recycling were unchanged in slices from methadone-treated animals. Remarkably the reduced recovery from desensitization and receptor recycling found in slices from morphine-treated animals were not observed in animals lacking ß-arrestin-2. Furthermore, pharmacological inhibition of G-protein receptor kinase 2 (GRK2), although not affecting the ability of [Met](5)enkephalin to induce desensitization, acutely reversed the delay in recovery from desensitization produced by chronic morphine treatment. These results characterize a previously unidentified function of the GRK/arrestin system in mediating opioid regulation in response to chronic morphine administration. They also suggest that the GRK/arrestin system, rather than serving as a primary mediator of acute desensitization, controls recovery from desensitization by regulating receptor reinsertion to the plasma membrane after chronic treatment with morphine. The sustained GRK/arrestin-dependent desensitization is another way in which morphine and methadone are distinguished.

Chemokine-receptor interactions: GPCRs, glycosaminoglycans and viral chemokine binding proteins.

A key feature of the immune system is the migration of leukocytes throughout the organism in an effort to patrol for infectious pathogens, tissue damage, and other physiological insults. This remarkable surveillance system is controlled by a family of proteins called chemokines (chemoattractant cytokines), and their respective receptors. Originally discovered because of their role in cell recruitment during inflammation, it is now well recognized that chemokines are also involved in other diverse processes including lymphocyte development and homing, organogenesis, and neuronal communication. While chemokines have evolved largely for host protection, their ability to induce cell damage and inappropriate cell recruitment, can lead to disease. Thus, there is considerable interest in developing antagonists. In this review we emphasize what is known about the structural biology of chemokines, chemokine receptors, and interactions with cell surface glycosaminoglycans. We also briefly describe their role in certain diseases and strategies for interfering with chemokine function that have emerged from mechanistic and structural understanding of their function. Finally we discuss viral mechanisms for sabotaging or manipulating the chemokine system, in part to illustrate the level of molecular mimicry that viruses have achieved and the evolutionary pressure imposed on the immune system by these pathogens.

Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

Examination of MCP-1 (CCL2) partitioning and presentation during transendothelial leukocyte migration.

It is proposed that a chemokine concentration gradient promotes vectorial leukocyte migration across the vascular endothelium during inflammation. In this study, monocyte migration across a model endothelial monolayer was assessed at defined time-points after the addition of MCP-1 (CCL2). At each time-point transendothelial migration was quantified, medium from the apical and basal surface was collected for ELISA and monolayers were stained to detect both heparan sulfate and MCP-1. Statistically significant monocyte migration was observed within 60 min of chemokine addition to the basal surface of the endothelium and an asymmetric distribution of MCP-1 across the monolayer was observed at all time-points. Dual color immunofluorescence analysis demonstrated that MCP-1 was focused into heparan sulfate-containing domains on the apical surface of some of the endothelial cells. Furthermore, no uniform concentration gradient of chemokine was observed within the space between adjacent endothelial cells with apical MCP-1 application resulting in a staining pattern identical to that observed after basal application. The addition of a functional, monomeric form of MCP-1 produced a staining pattern identical to that observed using the wild-type protein, suggesting that localized chemokine oligomerization is not responsible for generating the focal chemokine distribution. Together, these data suggest that apical presentation of concentrated, chemokine-containing domains provides sufficient stimulus to promote transendothelial leukocyte migration in the absence of the formation of a formal haptotactic concentration gradient between endothelial cells.

Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines.

During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seven-transmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1beta/CCL4, and RANTES/CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

Structural basis of chemokine sequestration by a herpesvirus decoy receptor.

The M3 protein encoded by murine gamma herpesvirus68 (gamma HV68) functions as an immune system saboteur by the engagement of chemoattractant cytokines, thereby altering host antiviral inflammatory responses. Here we report the crystal structures of M3 both alone and in complex with the CC chemokine MCP-1. M3 is a two-domain beta sandwich protein with a unique sequence and topology, forming a tightly packed anti-parallel dimer. The stoichiometry of the MCP-1:M3 complex is 2:2, with two monomeric chemokines embedded at distal ends of the preassociated M3 dimer. Conformational flexibility and electrostatic complementation are both used by M3 to achieve high-affinity and broad-spectrum chemokine engagement. M3 also employs structural mimicry to promiscuously sequester chemokines, engaging conservative structural elements associated with both chemokine homodimerization and binding to G protein-coupled receptors.