Homologous Desensitisation of the Mouse Leukotriene B₄ Receptor Involves Protein Kinase C-Mediated Phosphorylation of Serine 127

Abstract

Murine leukotriene B₄ (LTB₄) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB₄ receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB₄-evoked, Ca²⁺-activated Cl^- currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca²⁺-activated Cl^- current and LTB₄ concentration was demonstrated with an apparent EC₅₀ of 6.7 nM. Following LTB₄ stimulation of mBLT1, we observed two transient, spatially distinct Ca²⁺-activated, inwardly directed Cl^- currents in the oocytes: a fast peak current requiring relatively high LTB₄ concentrations, and a slowly progressing Cl^- current. Nucleotides, PGE₂, 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD₄ did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB₄-evoked Cl^- currents. Repetitive LTB₄ administration desensitized the LTB₄-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB₄-evoked currents, whereas down-regulation of PKC by prolonged PMA exposure (20 h) impaired mBLT1 desensitisation. In addition, Ser-127-Ala substitution of the PKC consensus phosphorylation site on the second intracellular loop prevented the mBLT1 desensitisation. These data indicate that PKC-mediated phosphorylation at Ser-127 leads to mBLT1 desensitisation.

FcγRI ligation leads to a complex with BLT1 in lipid rafts that enhances rat lung macrophage antimicrobial functions

“…44 We studied the role of phosphorylation in promoting both the association of BLT1 with Fc␥RI as well as the redistribution of both receptors to LRs. Whereas Fc␥RI engagement enhanced both serine and tyrosine phosphorylation of BLT1 in LRs, BLT1 redistribution to and physical association with Fc␥RI in LRs were not abrogated by the protein kinase C inhibitor calphostin C. Although serine/threonine phosphorylation of BLT1 has been implicated in receptor desensitization, 35,36 neither its tyrosine phosphorylation nor a role for its phosphorylation in downstream signaling events has been reported. In contrast to calphostin C, the broad spectrum PTK inhibitor genistein did abrogate the tyrosine phosphorylation of BLT1 as well as its redistribution to LRs and its interaction with the immunoreceptor.…”

“…Although serine 35 and threonine 36 phosphorylation of BLT1 have been implicated in receptor desensitization, neither its tyrosine phosphorylation nor a potential positive role for its phosphorylation in downstream signaling events has previously been investigated. Therefore, phosphorylation of BLT1 was examined by immunoblot analysis in pooled membrane fractions of BLT1 immunoprecipitates.…”

International Union of Basic and Clinical Pharmacology. LXXXIV: Leukotriene Receptor Nomenclature, Distribution, and Pathophysiological Functions

“…Through the substitution of all Ser and Thr residues in the C-terminal tail with Ala (to generate a phosphorylation-defective mutant) or with Asp/Glu (to mimic constitutive phosphorylation), it has been shown that BLT 1 R phosphorylation may be an important mediator of G protein activation, whereas ␤-arrestin-associated BLT 1 R internalization seems to be independent of phosphorylation (Jala et al, 2005). For the murine BLT 1 R, a PKC consensus phosphorylation site has been reported to be located on the second intracellular loop, and a Ser 127 substitution within this site prevented mBLT 1 R desensitization (Mollerup et al, 2007).…”

Double Mutation at the Putative Protein Kinase C Phosphorylation Sites Thr151Plus Thr323in the Mouse LeukotrieneD4Receptor Eliminates Homologous Desensitization

“…The two-electrode voltage clamp experiments were performed as previously described [40]. Briefly, we used a lab-made recording chamber and perfusion system, an OOC-1 amplifier (World Precision Instruments), a Digidata 1200B digital recording system, and AxoScope software (Axon Instruments Inc.) for sampling to a PC.…”

“…The equilibrium potentials for Cl - and K + in X. laevis oocytes are -21 mV and -101 mV, respectively (calculated from concentrations given in [41]. In order to avoid saturation of the amplifier during Ca 2+ -activated Cl - currents we used oocytes voltage clamped in the range +50 to -60 mV instead of -95 mV see [40]. …”