RESUMO
Opioid agonists have a broad range of effects on cells of the immune system, including modulation of the inflammatory response, and opioid and chemokine receptors are co-expressed by many white cells. Hetero-oligomerization of the human DOP opioid and chemokine CXCR2 receptors could be detected following their co-expression by each of co-immunoprecipitation, three different resonance energy transfer techniques and the construction of pairs of individually inactive but potentially complementary receptor G-protein alpha subunit fusion proteins. Although DOP receptor agonists and a CXCR2 antagonist had no inherent affinity for the alternative receptor when either receptor was expressed individually, use of cells that expressed a DOP opioid receptor construct constitutively, and in which expression of a CXCR2 receptor construct could be regulated, demonstrated that the CXCR2 antagonist enhanced the function of DOP receptor agonists only in the presence of CXCR2. This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy. Entirely equivalent results were obtained in cells in which the native DOP opioid receptor was expressed constitutively and in which expression of the isolated CXCR2 receptor could be induced. These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor. These results have novel and important implications for the development and use of small-molecule therapeutics.
Assuntos
Estrutura Quaternária de Proteína , Receptores de Interleucina-8B , Receptores Opioides delta/química , Receptores Opioides delta/metabolismo , Regulação Alostérica , Linhagem Celular , Dimerização , Diprenorfina/química , Diprenorfina/metabolismo , Leucina Encefalina-2-Alanina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/metabolismo , Compostos de Fenilureia/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Receptores Opioides delta/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A range of studies have indicated that many rhodopsin-like, family A G protein-coupled receptors, including the beta(2)-adrenoceptor, exist and probably function as dimers. It is less clear if receptors internalize as dimers and if agonist occupancy of only one element of a dimer is sufficient to cause internalization of a receptor dimer into the cell. We have used a chemogenomic approach to demonstrate that this is the case. Following expression of the wild type beta(2)-adrenoceptor, isoprenaline but not 1-(3''4'-dihydroxyphenyl)-3-methyl-1-butanone, which does not have significant affinity for the wild type receptor, caused receptor internalization. By contrast, 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone, but not isoprenaline that does not have high affinity for the mutated receptor, caused internalization of Asp(113)Serbeta(2)-adrenoceptor. Following co-expression of wild type and Asp(113)Serbeta(2)-adrenoceptors each of isoprenaline and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone caused the co-internalization of both of these two forms of the receptor. Co-expressed wild type and Asp(113)Serbeta(2)-adrenoceptors were able to be co-immunoprecipitated and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone produced internalization of the wild type receptor that was not prevented by the beta-adrenoceptor antagonist propranolol that binds with high affinity only to the wild type receptor. These results demonstrate that agonist occupancy of either single binding site of the beta(2)-adrenoceptor dimer is sufficient to cause internalization of the dimer and that antagonist occupation of one of the two ligand binding sites is unable to prevent agonist-mediated internalization.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacologia , Endocitose/efeitos dos fármacos , Linhagem Celular , Dimerização , Doxiciclina/farmacologia , Humanos , Isoproterenol/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas Mutantes/metabolismo , Propranolol/farmacologia , Proteínas do Envelope Viral/metabolismoRESUMO
Although traditionally assumed to be monomeric signaling units, G-protein-coupled receptors (GPCRs) have been shown to exist as dimers/oligomers. Many chemokine receptors have been demonstrated to form homo-oligomers, and hetero-oligomerization between both pairs of chemokine receptors and chemokine receptors and other GPCRs has also been demonstrated. This chapter highlights some of the most common techniques used to investigate chemokine receptor oligomerization.
Assuntos
Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoprecipitação , Medições Luminescentes , Multimerização ProteicaRESUMO
When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to beta-alanine but not L-alanine by elevating intracellular [Ca(2+)], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to beta-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of beta-alanine to phosphorylate ERK1 and ERK2 as well as maintaining the capacity of beta-alanine to elevate intracellular [Ca(2+)], which was reduced in magnitude and slowed in response with increasing times of expression of rMrgD in isolation. Associated with these effects, the presence of rMrgE restricted beta-alanine-induced internalization of rMrgD. This is the first report of heterodimeric interactions between members of the Mas-related gene (Mrg) receptor family and indicates that interactions between rMrgD and rMrgE modulate the function of rMrgD. Because the Mrg receptors are potential therapeutic targets in pain, these results suggest that efforts to understand the function and regulation of individual Mrg family receptors may require coexpression of relevant pairs.