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Structural insight into the role of the second intracellular loop of the bradykinin 2 receptor in signaling and internalization
Author(s) -
Piserchio Andrea,
Prado Gregory N.,
Zhang Ran,
Yu Jun,
Taylor Linda,
Polgar Peter,
Mierke Dale F.
Publication year - 2002
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.10072
Subject(s) - internalization , signal transduction , receptor , chemistry , phosphorylation , bradykinin receptor , g protein coupled receptor , mutant , serine , threonine , microbiology and biotechnology , biochemistry , intracellular , bradykinin , biophysics , biology , gene
The second cytoplasmic loop (IC2) of the bradykinin B2 receptor plays a vital role in its dynamic life cycle including the activation, internalization, desensitization, and resensitization of this receptor. Here, we probe the structure and function of the IC2, with particular emphasis on threonine‐137, which is crucial for signal transduction and internalization. Mutation of this threonine to proline (T137P) produces wild type (WT) signaling and complete inhibition of internalization. Incorporation of aspartate (T137D) leads to a marked reduction in receptor signaling but with WT receptor uptake. The T137D mutation coupled with serine to alanine substitution of S335 and S341 within the distal C‐terminus recovers signaling, leading to an actually enhanced arachidonic acid release and phosphoinositide turnover compared to WT bradykinin B2 receptor (BKB2R). To provide a structural basis for the actions of this mutant, the conformational features of IC2 (both WT and mutant) were investigated by high‐resolution NMR. The NMR analysis illustrated two prominent α‐helices at the N‐ (L123–M138) and C‐termini (A149–I156) of the IC2 receptor domain. Incorporating these structural characteristics into a model of BKB2R, we determined that the entire N‐terminal helix of IC2 is incorporated as TM3, placing Y131 1.5 helical turns into TM3 and T137 at the membrane surface. The NMR data indicated no structural changes upon substitution of T137D. These results suggest that the altered signaling of the T137D mutant can be attributed to the introduction of a negative charge, indicating that phosphorylation of this residue takes place and participates in the life cycle of this receptor. Additionally, the return to WT signal capacity of the mutation T137D/S335A/S341A, to overcome the deleterious T137D substitution points to a functional interaction between the IC2 and the C‐terminus. © 2002 John Wiley & Sons, Inc. Biopolymers 63: 239–246, 2002; DOI 10.1002/bip.10072