Ser-204 and Ser207 on transmembrane 5 (TM5) of the (2 adrenergic receptor ((2AR) are critical in interactions with catecholamines and other (2AR binding ligands. It is beleived that the hydroxyls of these tow amino acid residues form hydrogen bonds with (2AR agonists and antagonists. In this research project, a recombinant (2AR with Ser-204 to Cys (S204C) or S207C will be constructed, expressed in sf9 cells of baculovirus expression system. The purified receptor will be radiolabeled with thiocatecholamine derivatives (125I]-1-(1-oxy-3-hydroxy-4-thio-phenyl)-3-(N-iodo-tyramine)-2-pr opanol and (125I]-1-(1-oxy-3-thio-4-hydroxy-phenyl)-3-(N-iodo-tyramine)-2-pr opanol), which have a thio group attached to the catechol phenyl ring instead of the hydroxyl group. In addtion, novel photoactivable radioactive fluorenone and benzophenone derivatives have been synthesized for the binding site probing. The photoradiolabeled (2AR will be cleaved by proteases and the peptide mapping will be analyzed with polyclonal antibodies to TM5 using Western blotting techniques. By sequencing the protealytic peptides, the binding sites for those ligands can be identified by comparing the obtained sequence with the deduced amino acid sequence of the (2AR from the cDNA.
NIH ExPorter
NIH ExPorter