Neuroscience letters

The alkylating agent EEDQ facilitates protease-mediated degradation of the human brain alpha2A-adrenoceptor as revealed by a sequence-specific antibody.

PMID 10213146


An antibody against a sequence from the divergent third intracellular loop of the human alpha2A-adrenoceptor, amino acids 262-276, was produced. The antiserum was tested, along with the preimmune serum, by means of ELISA and dot blot assays which demonstrated that the alpha2A-peptide used for the antibody production was recognized by the immune serum. The antibody also recognized the alpha2A-adrenoceptor protein in the human brain (immunoblot analysis). In cortical membranes a major immunoreactive peptide of approximately 70 kDa (mature glycosylated receptor) was detected and after treatment with N-glycosidase F only a approximately 50 kDa peptide (non-glycosylated receptor) was immunodetected. This antibody was used to demonstrate that a chemical modification of the alpha2A-adrenoceptor induced by the alkylating agent EEDQ facilitates the protease-mediated receptor degradation. Thus in vitro, normal receptor degradation (24-44% at 2-4 h) was enhanced by EEDQ (10(-6) M) (38-71% at 2-4 h) but in the presence of protease inhibitors this effect was almost abolished.