SPECIFIC IMMUNOASSAY FOR THE BETA-2 ADRENERGIC RECEPTOR

THE BETA-2 ADRENERGIC RECEPTOR IS AN IMPORTANT NEUROTRANSMITTER RECEPTOR, WHICH IS INVOLVED IN THE REGULATION OF MULTIPLE CELLULAR SYSTEMS. DYSFUNCTION OF THERECEPTOR IS INVOLVED IN SEVERAL PATHOLOGICAL STATES INCLUDING ASTHMA, HYPERTENSION, MYOCARDITIS AND DILATED CARDIOMYOPATHY. MEASUREMENT OF BETA RECEPOR ACTIVITY RESTS PREDOMINANTLY ON THE BINDING ASSAY, WHICH HAS SEVERAL LIMITATIONS AND IS OF LIMITED CLINICAL UTILITY. THE GOAL IS TO PRODUCE SPECIFIC ANTIBODIES TO THE BETA ADRENERGIC RECEPTORS AND TO DEVELOP IMMUNOASSAYS USING THESE ANTIBODIES. TWO COMPLEMENTARY APPROACHES USING PROTEIN CHEMISTRY AND MOLECULAR BIOLOGY ARE DESCRIBED. IN THE FIRST APPROACH, ANTIGENIC REGIONS OF THE BETA ADRENERGIC RECEPTOR WILL BE IDENTIFIED USING THEORETICAL MODELS, PEPTIDES CORRESPONDING TO THESE SEQUENCES WILL BE SYNTHESIZED, ANTIBODIES PRODUCED IN RABBITS, AND THE ANTIBODIES EVALUATED FOR THE DETECTION OF THE DENATURED AND INTACT FORMS OF THE RECEPTOR. THE SECOND APPROACH INVOLVES EXPRESSING THE RECEPTOR IN E. COLI (AS A SECRETORY PROTEIN AND AS A BETA GALACTOSIDASE FUSION PROTEIN), IN A FORM WHICH RETAINS BINDING ACTIVITY AND PHARMACOLOGIC SPECIFICITY. THE EXPRESSED RECEPTOR WILL BE UTILIZED TO TEST THE ANTIBODIES RAISED WITH SYNTHETIC PEPTIDES, AS A SOURCE OF STANDARDS IN THE IMMUNOASSAYS, AND WILL ALSO SERVEAS THE ANTIGEN IN RAISING A FURTHER PANEL OF ANTIBODIES. SPECIFICITY OF THE ANTIBODIES WILL BE ASSESSED USING MEMBRANES DERIVED FROM DIFFERENT CELLS CONTAINING OR DEVOID OF THE BETA-2 RECEPTOR. FURTHER TESTS OF SPECIFICITY WILL ESTABLISH THE IMMUNOREACTIVE BAND ON WESTERN BLOTS AS BEING THE RECEPTOR BY CARRYING OUT IMMUNOPRECIPITATION OF THE PHOTOLABELED RECEPTOR. ONCE THE VALIDATION OF THE ANTIBODIES IS COMPLETE, A CLINICALLY USEFUL IMMUNOASSAY AS EIA WILL BE FORMULATED. THE ANTIBODIES WILL ALSO BE EVALUATED FOR THEIR UTILITY IN STRUCTURE/FUNCTION STUDIES OFTHE RECEPTOR. PRELIMINARY EVIDENCE IS GIVEN WHICH INDICATESTHAT ANTIBODIES TO SOME SYNTHETIC PEPTIDES MAY DETECT BOTH THE NATURAL AND RECOMBINANT RECEPTORS.