The alkylating agent EEDQ facilitates protease-mediated degradation of the human brain alpha2A-adrenoceptor as revealed by a sequence-specific antibody.
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.
Ozaita
A
A
Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain.
Escribá
P V
PV
García-Sevilla
J A
JA
D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000477:Alkylating Agents / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001921:Brain / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D011804:Quinolines / Q000494:pharmacology*; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*
C547768:ADRA2A protein, human; D000317:Adrenergic alpha-Antagonists; D000477:Alkylating Agents; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011804:Quinolines; D018341:Receptors, Adrenergic, alpha-2; C000470:EEDQ; D010450:Endopeptidases