Human kinin b! receptor: Ligand binding and functional characterization

The kinin B l receptor has been implicated in the pathogenesis of chronic inflammation and hyperalgesia. To further characterize and examine the mechanism of Bl responses, we studied the human Bl receptor human expressed as a stable clone in CHO cells. Binding studies were carried out for 1 hr at 4°C using intact cells in suspension with [3H]des-Argl 0 lysylbradykinin as a ligand. The Kd and Bmax were 0.33 ±0.05 nM and 37.3 ± 4.0 fmol/mg protein, respectively. The Ki values (nM) for kinin analogues in displacement experiments using 1 ,nM [3H]des-ArglO lysylbradykinin were as follows: bradykinin (BK), >10,000; lysylbradykinin (LBK), 88; des-ArglO LBK, 0.15; des-Arg9BK, 180; des-Arg!OLeu9 LBK, 0.66; des-Arg9Leu8 BK, 78; des-ArglO HOE 140, 11.8; Arg-BK, 925; Lys-LBK, 160; DLysBK, >10,000; DLys-des-ArglO LBK, 0.38; DLys-des-Arg9 BK, 29. All values are means from at least 3 experiments in duplicate. Stimulation of the cloned CHO cells expressing the human B l receptor with Des-ArglO LBK for 20 mins resulted in inositol phosphate generation, with an ED50 of 0.lOnM. In a single experiment, immunoprecipitation of photoaffinity labeled G proteins from membranes prepared from the same cells indicated specific coupling of the BI receptor to Gq and Gai 1/2. We conclude that the human Bl receptor can be expressed as a stable clone in CHO cells, that the presence of a Lys residue at position 1 of LBK and desArglO LBK analogs is critical for high affinity binding, and that the human Bl receptor is functionally coupled via a G-protein(s) to phospholipase C.