RESUMEN
The melanin-concentrating hormone (MCH) receptor type 1 (MCHR1) is a seven-transmembrane domain protein that modulates orexigenic activity of MCH, the corresponding endogenous peptide agonist. MCH antagonists are being explored as a potential treatment for obesity. In the current study, we examined the pharmacological impact of 11 naturally occurring mutations in the human MCHR1. Wild-type and mutant receptors were transiently expressed in human embryonic kidney 293 cells. MCHR1-mediated, Gα(i)-dependent signaling was monitored by using luciferase reporter gene assays. Two mutants, R210H and P377S, failed to respond to MCH. Five other variants showed significant alterations in MCH efficacy, ranging from 44 to 142% of the wild-type value. At each of the MCH-responsive mutants, agonist potency and inhibition by (S)-methyl 3-((3-(4-(3-acetamidophenyl)piperidin-1-yl)propyl)carbamoyl)-4-(3,4-difluorophenyl)-6-(methoxymethyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (SNAP-7941), an established MCHR1 small-molecule antagonist, were similar to wild type. To explore the basis for inactivity of the R210H and P377S mutants, we examined expression levels of these receptors. Assessment by enzyme-linked immunosorbent assay revealed that cell surface expression of both nonfunctional receptors was comparable with wild type. Overnight treatment with SNAP-7941, followed by washout of antagonist, enhanced MCH induced signaling by the wild-type receptor and restored MCH responsiveness of the P377S but not the R210H variant. It is of note that the two loss-of-function mutants were identified in markedly underweight individuals, raising the possibility that a lean phenotype may be linked to deficient MCHR1 signaling. Formal association studies with larger cohorts are needed to explore the extent to which signaling-deficient MCHR1 variants influence the maintenance of body weight.
Asunto(s)
Hormonas Hipotalámicas/farmacología , Melaninas/farmacología , Mutación Missense/fisiología , Hormonas Hipofisarias/farmacología , Polimorfismo de Nucleótido Simple/fisiología , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Genes Reporteros/genética , Células HEK293 , Humanos , Piperidinas/farmacología , Pirimidinas/farmacología , Receptores de Somatostatina/antagonistas & inhibidores , Receptores de Somatostatina/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Delgadez/genética , TransfecciónRESUMEN
The µ-opioid receptor (MOR) plays an important role in modulating analgesia, feeding behavior, and a range of autonomic functions. In the current study, we investigated the degree to which 13 naturally occurring missense mutations affect the pharmacological properties of the human MOR. After expression of each receptor in human embryonic kidney 293 cells, signaling (Gα(i/o)-mediated) induced by peptide agonists was assessed using luciferase reporter gene assays. Multiple mutants (S66F, S147C, R260H, R265C, R265H, and S268P) show a significant reduction in agonist potency. At the N190K variant, agonist-mediated signaling was essentially absent. Enzyme-linked immunosorbent assay, microscopic analysis, and radioligand binding assays revealed that this mutant shows markedly reduced cell-surface expression, whereas all other receptor variants were expressed at normal levels. Surface expression of the N190K variant could be increased by incubation with the alkaloid agonist buprenorphine or with either naltrexone or naloxone, structurally related MOR antagonists. We were surprised to find that both putative antagonists, despite being inactive at the wild-type MOR, triggered a concentration-dependent increase in N190K receptor-mediated signaling. In contrast, peptidic ligands failed to promote expression or rescue function of the N190K mutant. Subsequent analysis of the N190K variant in an ethnically diverse cohort identified this isoform in a subgroup of African Americans. Taken together, our studies reveal that the N190K mutation leads to severe functional alterations and, in parallel, changes the response to established MOR ligands. The extent to which this mutation results in physiological abnormalities or affects drug sensitivity in selected populations (e.g., those with chronic pain or addiction) remains to be investigated.
Asunto(s)
Péptidos/farmacología , Receptores Opioides mu/agonistas , Negro o Afroamericano , Sustitución de Aminoácidos , Línea Celular , HDL-Colesterol/sangre , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Femenino , Genes Reporteros , Genotipo , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Mutagénesis Sitio-Dirigida , Mutación Missense , Naloxona/farmacología , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Péptidos Opioides/farmacología , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Transporte de Proteínas , Ensayo de Unión Radioligante , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/genética , Transducción de Señal , Población BlancaRESUMEN
Preimplantation mouse embryos express both classical (class Ia) and nonclassical (class Ib) MHC class I proteins, and yet are not rejected by the maternal immune system. Although the function of the embryonic MHC class Ia proteins is unknown, one MHC class Ib protein, Qa-2, the product of the preimplantation embryo development (Ped) gene, actually enhances reproductive success. Similar in structure to MHC class Ia proteins, Qa-2 protein is a trimer of the alpha (heavy) chain, beta(2) microglobulin and a bound peptide. Studies on the folding, assembly and trafficking of MHC class Ia molecules to the cell surface have revealed this process to be dependent on multiple protein chaperone molecules, but information on the role of chaperone molecules in Qa-2 expression is incomplete. Here, we report the detection of mRNA for four chaperone molecules (TAP1, TAP2, calnexin and tapasin) in preimplantation embryos. We then focused on the role of the MHC-dedicated chaperone, tapasin, on Qa-2 protein expression. First, we demonstrated that tapasin protein is expressed by preimplantation embryos. Then, we used tapasin knockout mice to evaluate the role of tapasin in Qa-2 protein expression on both T cells and preimplantation embryos. We report here that optimal cell surface expression of Qa-2 is dependent on tapasin in both T cells and preimplantation embryos. Identification of the molecules involved in regulation of MHC class I protein expression in early embryos is an important first step in gaining insight into mechanisms of escape of embryos from destruction by the maternal immune system.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Blastocisto/inmunología , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Chaperonas Moleculares/inmunología , Linfocitos T/inmunología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Blastocisto/citología , Femenino , Regulación de la Expresión Génica/genética , Antígenos de Histocompatibilidad Clase I/genética , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Péptidos/inmunología , Embarazo , Pliegue de Proteína , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Linfocitos T/citología , Microglobulina beta-2/inmunologíaRESUMEN
Qa-2, a murine class Ib major histocompatibility complex (MHC) molecule, is a possible functional homolog of human leukocyte antigen G (HLA-G). Both molecules have been implicated in immunoregulation and embryonic development and both occur in membrane-bound and soluble isoforms that arise by alternative splicing. Soluble splice variants have been implicated in the reproductive functions of HLA-G. While soluble variants of Qa-2 have been previously detected in T lymphocytes, we now demonstrate the presence of mRNA for one of the two known soluble forms of Qa-2 in eight-cell embryos and in blastocysts. Qa-2 is glycosylphosphatidylinositol (GPI) linked in the outer leaflet of the cell membrane and is found in lipid raft microdomains where other raft-associated proteins transduce signals into the cell. In contrast, HLA-G has a truncated six amino acid cytoplasmic tail. By fluorescence co-localization in JEG-3 cells, using fluorescent cholera toxin beta subunit (a lipid raft marker) and anti-HLA-G antibody, we have demonstrated that membrane-bound HLA-G also localizes to lipid rafts, consistent with functional homology between the two molecules. Finally, our experiments in which we have purified Qa-2 and transferred it via a process known as protein painting to Qa-2 negative cells represent a model for potential therapy involving HLA-G.
Asunto(s)
Blastocisto/inmunología , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Animales , Línea Celular , Membrana Celular/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Antígenos HLA/análisis , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Microdominios de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Embarazo , ARN/aislamiento & purificación , Empalme del ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
The human major histocompatibility complex (MHC), in addition to its role in the regulation of cell-cell interactions in the immune response, also influences reproductive success. Human leukocyte antigen-G (HLA-G) is an MHC class I gene of particular interest in reproductive biology because of its specific expression on fetal cytotrophoblast cells, and its reported involvement both in protection of the developing fetus from destruction by the maternal immune response and in the prevention of maternal pre-eclampsia. HLA-G has 15 known alleles at the DNA level, and allelic frequency varies among ethnic groups. This study describes the results of an inaugural attempt to correlate an HLA-G genetic polymorphism with pregnancy outcome in a patient population undergoing IVF. The study group was composed of 102 Caucasian women. A maternal HLA-G genetic polymorphism was investigated by polymerase chain reaction (PCR) analysis of DNA collected from granulosa cells surrounding oocytes harvested for the IVF procedure. While no statistically significant correlation was identified in this initial study, larger studies examining DNA from trios of mother, father and offspring are planned.