Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Structural determinants regulating expression of the high affinity leukotriene B4 receptor: involvement of dileucine motifs and alpha-helix VIII.

Mutational analysis of determinants located in the C-terminal (C) tail of the high affinity leukotriene (LT) B(4) receptor, BLT1, was performed to assess their significance in BLT1 trafficking. When expressed in COS-7 cells, a BLT1 deletion mutant lacking the C-tail (G291stop) displayed higher numbers of binding sites and increased signal transduction compared with wild-type (WT) BLT1. Addition of the C-tail from either the platelet-activating factor receptor or the LTD(4) receptor, CysLT1, did not restore WT phenotype. Moreover, the number of LTB(4) binding sites was higher in the chimeras than in the WT BLT1, suggesting the requirement for specific structural determinants within the BLT1 C-tail. Elimination of a distal C-tail dileucine motif (Leu(304)-Leu(305)), but not the proximal (Leu(292)-Leu(293)) motif, altered BLT1 pharmacological characteristics and caused a moderate constitutive receptor activation. Surprisingly, all mutant receptors were efficiently delivered to the plasma membrane, but not to a greater extent than WT BLT1, as assessed by flow cytometry. Furthermore, substitution of Leu(304)-Leu(305) prevented LTB(4)-induced BLT1 internalization. Molecular modeling of BLT1 on the bovine rhodopsin receptor scaffold strongly suggested the involvement of the distal dileucine motif (Leu(304)-Leu(305)) in a hydrophobic core, including intrahelical interactions within alpha-helix VIII and interhelical interactions with residues of helix I. Disruption of this hydrophobic core is proposed to increase the population of receptors in the active form, to restrain their trafficking and to facilitate the activation of BLT1 as indicated by the increased maximal level of binding of the ligand and constitutive activation of the receptor.

Threonine 308 within a putative casein kinase 2 site of the cytoplasmic tail of leukotriene B(4) receptor (BLT1) is crucial for ligand-induced, G-protein-coupled receptor-specific kinase 6-mediated desensitization.

Desensitization of G-protein-coupled receptors may involve phosphorylation of serine and threonine residues. The leukotriene B(4) (LTB(4)) receptor (BLT1) contains 14 intracellular serines and threonines, 8 of which are part of consensus target sequences for protein kinase C (PKC) or casein kinase 2. In this study, we investigated the importance of PKC and GPCR-specific kinase (GRK) phosphorylation in BLT1 desensitization. Pretreatment of BLT1-transfected COS-7 cells with PKC activators caused a decrease of LTB(4)-induced inositol phosphate (IP) accumulation. This reduction was prevented with the PKC inhibitor, staurosporine, and not observed in cells expressing a BLT1 deletion mutant (G291stop) lacking the cytoplasmic tail. Moreover LTB(4)-induced IP accumulation was significantly inhibited by overexpression of GRK2, GRK5, and especially GRK6, in cells expressing wild type BLT1 but not in those expressing G291stop. GRK6-mediated desensitization correlated with increased phosphorylation of BLT1. The G319stop truncated BLT1 mutant displayed functional characteristics comparable with wild type BLT1 in terms of desensitization by GRK6, but not by PKC. Substitution of Thr(308) within a putative casein kinase 2 site to proline or alanine in the full-length BLT1 receptor prevented most of GRK6-mediated inhibition of LTB(4)-induced IP production but only partially affected LTB(4)-induced BLT1 phosphorylation. Our findings thus suggest that Thr(308) is a major residue involved in GRK6-mediated desensitization of BLT1 signaling.