Coulombic and Hydrophobic Interactions in the First Intracellular Loop Are Vital for Bradykinin B2 Receptor Ligand Binding and Consequent Signal Transduction

Abstract

The role of the first intracellular loop (IC1) in the function of the rat bradykinin B2 receptor (BKB2R) was probed. On the basis of the bovine rhodopsin X-ray structure, the BKB2R IC1 consists of six residues: (60)HKTNCT. Exchange of this sequence with the bradykinin B1 receptor IC1 (PRRQLN) resulted in a chimera which bound bradykinin and signaled as wild-type (WT) BKB2R. In contrast, a chimera containing the IC1 of rat angiotensin II type Ia receptor (AT1aR) (YMKLKT) did not bind BK nor signal in response to BK at a concentration as high as 5 microM. ELISA illustrated that this receptor was still processed and inserted into the plasma membrane. Employing portions of the IC1, we observed that (60)HKT of BKB2R could be exchanged as a group with either the BKB1R (PRR) or AT1aR (YMK) with no change in receptor binding or signaling activities. When only the YM of AT1aR replaced the HK of BKB2R, leaving the N-terminal portion of IC1 without a positively charged residue, binding and signaling were reduced by more than 70%. When only N63 was replaced with the corresponding leucine of AT1aR, binding and signaling were ablated. In fact, replacement of the entire IC1 with the AT1aR except for N63 resulted in binding and signaling as WT BKB2R. However, N63 could be replaced by glutamine (in BKB1R) or aspartate and continued to function as WT BKB2R. NMR data indicated that the BKB2R IC1 extends beyond the bovine rhodopsin prototype to include HKTNCTVAEI. When E68 was exchanged with a serine (in AT1aR), ligand binding decreased by 60% and PI turnover decreased by 69%. Molecular modeling points to a strict requirement for a hydrophilic residue at position 63 (N) at the middle of the IC1 and a Coulombic charge interaction between the positive charges (H60 and K61) at the N-terminus and a negative charge (E68) at the C-terminus of the IC1.

Activation of ERK, JNK, Akt, and G‐protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK‐293 cells

“…Previously we have found that both the AT1 and BKB2 receptors contain certain sequences within their cytosolic faces which are irreplaceable with respect to binding of the ligand or interaction with Gαi and Gαq. For example, in BKB2R the IC1 has to contain N63 to continue to bind [Yu et al, 2005]. In the proximal tail BKB2R has to contain KSRE to continue to signal with Gαi and Gαq [Yu et al, 2004].…”

“…Cell culture and transfections in HEK‐293 cells were performed as described by Yu et al 2005. HEK‐293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin at 37°C in a humidified CO 2 (5%) incubator.…”

“…Receptor binding studies of the BKB2R, AT1R, and mutant receptors in intact HEK293 cells were carried out as described by Yu et al 2005. Briefly, confluent cell monolayers in 24‐well plates were incubated in binding buffer (50 mmol/L Tris, 120 mmol/L NaCl, 4 mmol/L KCl, 10 µg/ml bacitracin, 10 mmol/L glucose, 0.1% BSA, 1 mmol/L CaCl 2 , 5 mmol/L MgCl 2 , 10 mmol/L HEPES pH 7.35) containing various concentrations of [ 3 H]‐AngII ranging from 0.04 to 10 nM in the absence (total binding) or presence of 100 nM unlabeled AngII (nonspecific binding) for 2 h at 4°C.…”

“…The generation of the molecular models has been previously described [Yu et al, 2005]. The receptor hybrids were created using the MSI Insight II program (Accelrys®) and then soaked in a two‐phase simulation cell of water/decane.…”

Attenuation of Angiotensin II-Induced Hypertension and Cardiac Hypertrophy in Transgenic Mice Overexpressing a Type 1 Receptor Mutant

“…AngII binding studies in intact aortic endothelial cells were carried out as described by Yu et al 11.…”