Role of hydroxyl containing residues in the intracellular region of rat bradykinin B-2 receptor in signal transduction, receptor internalization, andresensitization

In past reports we illustrated the importance of Y131, Y322, and T137 withi n the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) fo r signal transduction and receptor maintenance (Prado et al. [1997] J. Biol . Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555 ). In this report, we mutate the remaining hydroxyl possessing residues loc ated within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) di d not affect BK activated phosphatidylinositol (PI) turnover or receptor in ternalization. Chimeric exchange of the last 34 amino acids of BKB2R C-term inus with the corresponding 34 amino acids of the rat angiotensin II AT1 a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant wit h normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR f ragment (326-342), resulted in a receptor with a retarded internalization b ut a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V s howed little change in receptor uptake but a pronounced loss of BK activate d PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in v ery poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S34 8A led to poor internalization; whereas the retention of S348 and mutation of S341 A resulted in a receptor with a much greater internalization than W T. These and other results suggest that the presence of S348 promotes inter nalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate th at the distal C-terminus including its S/T cluster is important for both rB KB2R internalization and signal transduction. Individual S/T residues withi n this cluster appear involved in either signal transmission or receptor up take capacity. However, replacement of the entire distal tail region with t he corresponding rAT1aR sequence, also containing an S/T cluster, enables t he BKB2R/AT1aR chimera to act in a very similar manner to wild type rBK82R. J. Cell. Biochem. 83: 435-447, 2001. (C) 2001 Wiley-Liss, Inc.