title: 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 (Tyr131 and Tyr322) to signal transduction and receptor internalization (Prado, G. N., Taylor, L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638-14642). Here, we investigate the effect of exchanging both tyrosine residues simultaneously for alanine, phenylalanine, or serine, termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S) receptors, respectively. All of these mutants bound bradykinin (BK) normally, with a Kd of approximately 1.1 nM. However, although phosphoinositide (PI) turnover in response to BK by Y131A and Y131S proved negligible, the YAYA mutant returned BK-activated PI turnover to wild type (WT). In contrast, PI turnover with YSYS remained unresponsive to BK. Importantly, the pattern of BK-activated arachidonate release differed markedly in the mutant receptors. For example, whereas Y131S ablated BK-activated arachidonic acid release, conversion of this mutant to YSYS returned the BK-activated receptor function to a level above that of WT. However, YAYA showed only a partial recovery from the poor BK response of Y131A. These and additional results suggest that Tyr131 and Tyr322 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 Thr137 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 Thr137 is functioning as a switch in termination of signal transduction and the initiation of internalization.
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
authors.forename: G N
authors.initials: GN
authors.lastname: Prado
authors.forename: D F
authors.initials: DF
authors.lastname: Mierke
authors.forename: M
authors.initials: M
authors.lastname: Pellegrini
authors.forename: L
authors.initials: L
authors.lastname: Taylor
authors.forename: P
authors.initials: P
authors.lastname: Polgar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002460:Cell Line; D004705:Endocytosis / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D010716:Phosphatidylinositols; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D016718:Arachidonic Acid; D014443:Tyrosine