RESUMEN
The initially orphan human calcitonin (CT) receptor-like receptor (hCRLR) interacts with novel accessory receptor activity-modifying protein 1 (RAMP1) to reveal a functional CT gene-related peptide (CGRP) receptor. In mammalian cells, RAMP1 is required for mature N-glycosylation of the hCRLR predicted to occur at Asn(60), Asn(112), and/or Asn(117) in the amino-terminal extracellular domain. Here we have shown that the substitution of Asn(117) with Ala, Gln, Thr, or Pro abolished CGRP-evoked cAMP formation which was left unchanged when the Asn(117) was replaced with Asp. Moreover, the hCRLR and the Asn(117) mutants exhibited comparable N-glycosylation and cell surface expression, and the association with RAMP1 was only slightly impaired. In contrast, the hCRLR Asn(60,112) to Thr double mutant exhibited defective RAMP1-dependent N-glycosylation, and impaired cell surface expression and CGRP receptor function. Unlike Asn(60) and Asn(112), Asn(117) is normally not N-glycosylated, but essential for CGRP binding to the hCRLR-RAMP1 complex.
Asunto(s)
Asparagina/genética , Proteínas de la Membrana/fisiología , Mutagénesis Sitio-Dirigida , Receptores de Péptido Relacionado con el Gen de Calcitonina/genética , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Sustitución de Aminoácidos/genética , Animales , Células COS , Proteína Similar al Receptor de Calcitonina , Línea Celular Transformada , Membrana Celular/genética , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , AMP Cíclico/biosíntesis , Vectores Genéticos , Glicosilación , Humanos , Péptidos y Proteínas de Señalización Intracelular , Radioisótopos de Yodo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Pruebas de Precipitina , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteína 1 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Calcitonina/biosíntesis , Receptores de Calcitonina/genética , Receptores de Calcitonina/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/deficiencia , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
The human calcitonin (CT) receptor-like receptor (hCRLR) of the B family of G protein-coupled receptors is N-glycosylated and associates with receptor-activity-modifying proteins for functional interaction with CT gene-related peptide (CGRP) or adrenomedullin (ADM), respectively. Three putative N-glycosylation sites Asn(60), Asn(112) and Asn(117) are present in the amino-terminal extracellular domain of the hCRLR. Tunicamycin dose-dependently inhibited the glycosylation of a myc-tagged hCRLR and in parallel specific [(125)I]CGRP and -ADM binding. Similarly, the double mutant myc-hCRLR(N60,112T) exhibited minimal N-glycosidase F sensitive glycosylation, presumably at the third Asn(117), and the cell surface expression and specific radioligand binding were impaired. Substitution of the Asn(117) by Thr abolished CGRP and ADM binding in the face of intact N-glycosylation and cell surface expression.
Asunto(s)
Membrana Celular/metabolismo , Receptores de Calcitonina/metabolismo , Adrenomedulina , Amidohidrolasas/metabolismo , Asparagina/genética , Asparagina/metabolismo , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Western Blotting , Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteína Similar al Receptor de Calcitonina , Células Cultivadas , Relación Dosis-Respuesta a Droga , Glicosilación/efectos de los fármacos , Humanos , Mutagénesis Sitio-Dirigida , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ensayo de Unión Radioligante , Receptores de Calcitonina/genética , Tunicamicina/farmacologíaRESUMEN
Receptor-activity-modifying proteins (RAMPs) with single transmembrane domains define the function of two G-protein-coupled receptors of the B family. Cell-surface complexes of human RAMP1 (hRAMP1) and human calcitonin (CT) receptor isotype 2 (hCTR2) or rat CT-receptor-like receptor (rCRLR) have now been identified through protein cross-linking, co-immunoprecipitation and confocal microscopy. They are two distinct CT-gene-related peptide (CGRP) receptors coupled to cAMP production and pharmacologically distinguished by the CT and CGRP antagonists salmon CT(8-32) and human or rat CGRP(8-37). Thus direct molecular interactions of hRAMP1 with hCTR2 or rCRLR are required for CGRP recognition. hCTR2, moreover, adopts non-traditional functions through its association with hRAMP1.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Western Blotting , Células COS , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/farmacología , AMP Cíclico/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Microscopía Confocal , Pruebas de Precipitina , Unión Proteica , Ratas , Proteína 1 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Calcitonina/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , TransfecciónRESUMEN
Differential glycosylation of human and rat (r) calcitonin (CT) receptor-like receptors (CRLR) as a result of interactions with accessory receptor activity-modifying proteins (RAMP)1 or -2 was considered to define CT gene-related peptide (CGRP) or adrenomedullin (ADM) receptors in mammalian cells. Here, Drosophila Schneider (S2) cells stably co-expressed rCRLR and RAMP1 or -2 as functional CGRP or ADM receptors. Different from mammalian cells, rCRLR expressed in S2 cells are uniformly glycosylated proteins independent of RAMP1 or RAMP2. Bis(sulfosuccinimidyl)suberate cross-linking revealed receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMPs and [(125)I]CGRP or [(125)I]ADM. In conclusion, [(125)I]CGRP/rCRLR/RAMP1 and [(125)I]ADM/rCRLR/RAMP2 complexes have been recognized in Drosophila S2 cells.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Drosophila melanogaster/citología , Proteínas de la Membrana/metabolismo , Receptores de Calcitonina/metabolismo , Receptores de Péptidos/metabolismo , Adrenomedulina , Amidohidrolasas/metabolismo , Animales , Western Blotting , Péptido Relacionado con Gen de Calcitonina/antagonistas & inhibidores , Péptido Relacionado con Gen de Calcitonina/química , Proteína Similar al Receptor de Calcitonina , Línea Celular , Reactivos de Enlaces Cruzados/metabolismo , AMP Cíclico/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Glicosilación , Humanos , Concentración 50 Inhibidora , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Peso Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Péptidos/antagonistas & inhibidores , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Ratas , Proteína 1 Modificadora de la Actividad de Receptores , Proteína 2 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Adrenomedulina , Receptores de Calcitonina/química , Receptores de Calcitonina/genética , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Succinimidas/metabolismo , TransfecciónRESUMEN
Cortex Betulae contains (+)-catechin-7-beta-D-xylopyranoside. Except for its occurrence in elm bark, this biologically active substance is so far unknown as a natural product.
