Motif Mutation of Bradykinin B2 Receptor Second Intracellular Loop and Proximal C Terminus Is Critical for Signal Transduction, Internalization, and Resensitization

Abstract

In the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr 137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr 137 is functioning as a switch in termination of signal transduction and the initiation of internalization.The bradykinin B2 receptor (BKB2R) 1 cDNA has been successfully transfected into such cell types as hamster lung fibroblasts, Rat-1 fibroblasts and CHO cells with the expected binding properties (1-3). The BKB2R is a typical G protein-coupled receptor (GPCR) that has been reported to be associated with Gq (4 -7), Gi, and G 12 (8 -10). Binding of a GPCR to more than one G␣ subunit is not unprecedented. For example, the angiotensin II type 1 (AT1) receptor couples to both Gi and Gq (11). Whereas Gq has been linked to phospholipase C activation (6, 7), the release of arachidonate, via phospholipase A2 activation, has been linked to .The cellular response of GPCRs to agonists is regulated under a tightly controlled process of desensitization and resensitization (15, 16). Desensitization prevents cells from uncontrolled stimulation, and resensitization allows cells to recover and maintain responsiveness (17). Both processes appear to be regulated at the receptor level. Recent studies by Haasemann et al. (18) showed that stimulation of BKB2R results in redistribution of the receptor and its internalization in caveolae (18). On/off regulatory elements have been identified in other receptors (19 -22). However, to date, no clear consensus motifs have been identified to determine either internalization or resensitization. The DRYXXI/VXXP motif located in the second intracellular loop (IC2) of the human muscarinic cholinergic receptor has been proposed as important for receptor internalization (23). Another important motif is the tyrosine-containing motif located in the proximal BKB2R C terminus. The motifs in this region have been implicated in receptor internalization in other GPCRs, such as neurokinin 1 receptor (1...

Interaction of Calmodulin with the Serotonin 5-Hydroxytryptamine2A Receptor

“…However, these regions have previously been implicated in numerous signaling pathways and processes. The importance of the second intracellular loop in G protein coupling has been experimentally confirmed for other GPCRs, including the bradykinin B 2 (62), ␣ 2A adrenergic (63), and type B endothelin receptors (64). Using molecular modeling, Prado et al (62) produced evidence that the distal i2 loop and proximal carboxyl terminus of the bradykinin B 2 receptor may interact with each other to mediate receptor internalization and resensitization, in addition to G protein coupling.…”

“…The importance of the second intracellular loop in G protein coupling has been experimentally confirmed for other GPCRs, including the bradykinin B 2 (62), ␣ 2A adrenergic (63), and type B endothelin receptors (64). Using molecular modeling, Prado et al (62) produced evidence that the distal i2 loop and proximal carboxyl terminus of the bradykinin B 2 receptor may interact with each other to mediate receptor internalization and resensitization, in addition to G protein coupling. Berg et al (65) showed that the i2 loop of the 5-HT 2C receptor is important for G␣ q coupling, and that different isoforms of the receptor produced via RNA editing in regions of the second intracellular loop varied in their ability to induce second messengers.…”

Molecular Characterization of the Substance P·Neurokinin-1 Receptor Complex

“…The molecular model of the human NK-1R was built using the topological arrangement of the transmembrane helices of rhodopsin (18 -20) following procedures detailed previously (21,22). The loops connecting the transmembrane helices were generated using a metric matrix distance geometry program, employing the distances between the transmembrane helices as restraints.…”

“…The model contains the structural features of the second extracellular loop (EC2) of the NK-1R as recently determined by high resolution NMR (13). The loop plays an important role in SP binding, as evidenced by previous photoaffinity studies (13)(14)(15)(16)(17) a covalent bond with a residue in the proximal N terminus of the NK-1R (residues [11][12][13][14][15][16][17][18][19][20][21]. The structural consequences of Bpa 3 SP interacting with two sites, either sequentially or simultaneously, are examined here by extensive molecular dynamics simulations.…”

Ace inhibitors - activators of kinin receptors

“…С другой стороны, эта мутация полностью восстанавливала сопряжение рецептора с G-белком, которое уничтожалось при замене остатка Tyr 131 (Tyr 129 в Б 2 Р человека) входящего в состав DRY-мотива (II внутриклеточная петля). Различные дополнительные мутации аминокислотных остатков привели к выводу, что аминокислотные остатки тирозина вовлечены во взаимодействие, которое контролируется остатком Tyr 137 (Tyr 135 в Б 2 Р человека), что является очень важным для активации сопряжения c G белком и последующей интернализации рецептора [77].…”