Kinins Promote B₂Receptor Endocytosis and Delay Constitutive B₁Receptor Endocytosis

Abstract

Upon sustained insult, kinins are released and many kinin responses, such as inflammatory pain, adapt from a B 2 receptor (B 2 R) type in the acute phase to a B 1 receptor (B 1 R) type in the chronic phase. In this study, we show that kinins modulate receptor endocytosis to rapidly decrease B 2 R and increase B 1 R on the cell surface. B 2 Rs, which require agonist for activity, are stable plasma membrane components without agonist but recruit ␤-arrestin 2, internalize in a clathrin-dependent manner, and recycle rapidly upon agonist treatment. In contrast, B 1 Rs, which are inducible and constitutively active, constitutively internalize without agonist via a clathrin-dependent pathway, do not recruit ␤-arrestin 2, bind G protein-coupled receptor sorting protein, and target lysosomes for degradation. Agonist delays B 1 R endocytosis, thus transiently stabilizing the receptor. Most of the receptor trafficking phenotypes are transplantable from one receptor to the other through exchange of the C-terminal receptor tails, indicating that the tails contain epitopes that are important for the binding of protein partners that participate in the endocytic and postendocytic receptor choices. It is noteworthy that the agonist delay of B 1 R endocytosis is not transplanted to the B 2 R via the B 1 R tail, suggesting that this property of the B 1 R requires another domain. These events provide a rapid kinin-dependent mechanism for 1) regulating the constitutive B 1 R activity and 2) shifting the balance of accessible receptors in favor of B 1 R.

Kinin B1 receptor homo-oligomerization is required for receptor trafficking to the cell surface

“…2A). Live FB1 staining also revealed distinct intracellular puncta, which represents spontaneous receptor endocytosis, as described previously [5]. On the other hand, HB1stop135 expressed alone was localized exclusively intracellularly in a tubular network ( Fig.…”

“…Under these conditions, the intracellular puncta observed in fixed FB1 staining (Fig. 2B) do not represent constitutive receptor endocytosis because they still appeared when live staining was preceded by hyperosmotic sucrose treatment of the cells (data not shown), which blocks spontaneous receptor endocytosis [5]. HB1stop135 remained intracellular in the presence of FB1, and the two proteins partially co-localized in distinct puncta (Fig.…”

“…Human B1R and B2R cDNAs were subcloned into a pcDNA3.1 vector containing a zeocin or hygromycin selection marker. An artificial signal sequence and the FLAG tag were added in series to make FLAG-tagged B1R (FB1) and B2R (FB2) as described previously [5]. To make HA-tagged B1R (HB1), HA-tagged B1stop135 (HB1stop135), and HAtagged B2R (HB2), receptor cDNA in pcDNA3.0, containing a G418 selection marker, was mutated using a polymerase chain reaction-ligation-polymerase chain reaction protocol and the HA epitope was inserted at the receptor N terminus as described previously [19,20].…”

“…B2R is constitutively and ubiquitously expressed, signals in response to bradykinin (BK) and Lys-BK [1,2], rapidly desensitizes and internalizes upon agonist binding, and recycles to the plasma membrane following internalization [3][4][5][6]. In contrast, B1R expression is induced by sustained insult [1,2], signals in response to the carboxypeptidase products desArg 9 -BK and Lys-desArg 9 -BK [1,2], slowly desensitizes upon agonist binding [3], internalizes constitutively [5,7] in a manner that is impeded by the agonist [5], and the internalized receptor targets lysosomes for degradation [5].…”

“…B1R is constitutively active, at least in recombinant HEK293 cells [13], which may be detrimental under healthy conditions. Recent studies have shown that a relatively large portion of the cellular B1R pool in HEK293 cells is intracellular, in part localized in the endoplasmatic reticulum (ER), with relatively few receptors at the cell surface [5,14]. Indeed, B1R exits ER slowly and is subject to significant degradation via ER-associated degradation (ERAD) [14].…”