Bradykinin-induced Internalization of the Human B2Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail

Abstract

Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the liganddependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of ␤-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.

Antagonist-Induced Intracellular Sequestration of Rabbit Bradykinin B 2 Receptor

“…Finally, BK or the peptide antagonists translocated a fraction of the immunoreactive B 2 R-GFP to a dense cellular fraction containing endosomes and lysosomes (Figure 5B), 13 consistent with the recent finding of agonist-induced redistribution of B 2 Rs into caveolae 17 and internalization. 12 Again, LF 16.0687 was not active in this respect, consistent with the fact that functional receptors were recovered when this drug was washed away ( Figure 1 and Figure 2C). …”

“…The discrepancy may originate from a loss of immunoreactivity of the internalized B 2 R in rabbit tissues or from a saturation of the receptor breakdown mechanisms in HEK 293 cells expressing B 2 R-GFP at high levels. Heterologously expressed B 2 R-GFP is a suitable system to test various hypotheses about the mechanism of antagonist-induced sequestration, which may involve a partial agonist behavior of icatibant or NPC 17731 (although not detected in any of the functional assays applied), phosphorylation and internalization pathways common to those recruited by agonists, 12 or a different and novel mechanism. NPC 17731 may be less effective than icatibant in this respect ( Figure 5B).…”

“…In the present study, we investigated the competitive nature and reversibility of a constrained peptide structurally related to icatibant (NPC 17731, D-Arg[Hyp 3 , D-HypE(transpropyl) 7 , Oic 8 ]-BK) 11 and of a novel nonpeptide antagonist (LF 16.0687) 6 in the rabbit jugular vein; the behavior of B 2 R antagonists was correlated with events posterior to receptor binding in this species, with comparison to the wellestablished agonist-induced internalization of the B 2 R. 12 …”

Local Bradykinin Formation Is Controlled by Glycosaminoglycans

“…Notably, the effects of bradykinin are regulated on at least three levels: first, bradykinin generation is controlled by the assembly, activation, and dissociation of binary complexes of HK with PK (7,33); second, bradykinin signaling is regulated through specific B 2 Rs that rapidly desensitize and internalize upon bradykinin stimulation (40,52,53); and third, bradykinin degradation is efficiently executed by peptidases such as angiotensin-converting enzyme (54 -56). Loss of control over any of these levels may have severe pathophysiological consequences.…”

Ace inhibitors - activators of kinin receptors

“…В последнее десятилетие в рецепторах кининов были идентифицированы аминокислотные остатки, важные для взаимодействия с G-белком и дальнейшей передачи сигнала. Было показано, что усечение С-концевой последовательности в Б 2 Р человека вплоть до Ser 316 ослабляет, но полностью не тормозит сопряжение рецептора с Ga q -белком [74][75][76]. С другой стороны, усечение выше этого аминокислотного остатка полностью уничтожает активность Б 2 Р, что возможно приводит к разрыву a-cпирали (a8), включающей аминокислотные остатки 310 -321, которая, по-видимому, требуется для эффективного сопряжения рецептора с Ga q -белком (рис.…”