Ser204 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 believed that the hydroxyls of these two amino acid residues form hydrogen bonds with ?2AR agonists and antagonists. In this research project, a recombinant ?2AR with Ser204 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-propanol and ([125I]-1-(1-oxy-3-thio-4-hydroxy-phenyl)-3-(N-iodo-tyramine)-2-propanol which have a thio group attached to the catechol phenyl ring instead of a hydroxyl group. In addition, 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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
2P41RR002301-16
Application #
6309215
Study Section
Project Start
2000-04-15
Project End
2005-02-28
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
16
Fiscal Year
2000
Total Cost
$7,533
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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