RESUMO
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.
Assuntos
Asparagina/genética , Proteínas de Membrana/fisiologia , Mutagênese Sítio-Dirigida , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Substituição de Aminoácidos/genética , Animais , Células COS , Proteína Semelhante a Receptor de Calcitonina , Linhagem Celular Transformada , Membrana Celular/genética , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , AMP Cíclico/biossíntese , Vetores Genéticos , Glicosilação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Radioisótopos do Iodo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Testes de Precipitina , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteína 1 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/biossíntese , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/deficiência , Proteínas Recombinantes/biossíntese , TransfecçãoRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Receptores da Calcitonina/metabolismo , Adrenomedulina , Amidoidrolases/metabolismo , Asparagina/genética , Asparagina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/genética , Western Blotting , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina , Células Cultivadas , Relação Dose-Resposta a Droga , Glicosilação/efeitos dos fármacos , Humanos , Mutagênese Sítio-Dirigida , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ensaio Radioligante , Receptores da Calcitonina/genética , Tunicamicina/farmacologiaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Animais , Western Blotting , Células COS , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , AMP Cíclico/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Microscopia Confocal , Testes de Precipitina , Ligação Proteica , Ratos , Proteína 1 Modificadora da Atividade de Receptores , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , TransfecçãoRESUMO
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.
