STABLE EXPRESSION OF THE HUMAN KININ B-1 RECEPTOR IN CHINESE-HAMSTER OVARY CELLS - CHARACTERIZATION OF LIGAND-BINDING AND EFFECTOR PATHWAYS

To delineate ligand binding and functional characteristics of the huma n B-1 kinin receptor, a stable clone of Chinese hamster ovary cells ex pressing a single class of binding sites for [H-3] des-Arg(10)-lysylbr adykinin with a K-d of 0.3 mM and a B-max of 38 fmol/mg protein (simil ar to 40,000 receptors/cell) was isolated. Studies with peptide analog s showed that a lysine residue at position 1 (based on the lysylbradyk inin sequence) of ligands was essential for high affinity binding to t he human B-1 receptor. In marked contrast to cloned Chinese hamster ov ary cells expressing the human kinin B-2 receptor, which internalized approximately 80% of the ligand within 5 min upon exposure to 2 nM [H- 3]bradykinin, exposure of cells expressing the B-1 receptor to 1 nM [H -3] des-Arg(10)-lysylbradykinin resulted in minimal ligand internaliza tion. Stimulation of the B-1 receptor led to inositol phosphate genera tion and transient increases in intracellular calcium, confirming coup ling to phospholipase C, while immunoprecipitation of photoaffinity-la beled G-proteins from membranes indicated specific coupling of the rec eptor to G alpha q/11 and G alpha i(1,2). The B-1, unlike the B-2, rec eptor does not desensitize (as demonstrated by continuous phosphoinosi tide hydrolysis), enhancing the potential role of this receptor during inflammatory events.