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.
Gaudreau
Rémi
R
Immunology Division, Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke, Quebec J1H 5N4, Canada.
Beaulieu
Marie-Eve
ME
Chen
Zhangguo
Z
Le Gouill
Christian
C
Lavigne
Pierre
P
Stanková
Jana
J
Rola-Pleszczynski
Marek
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018102:Receptors, Leukotriene B4 / Q000096:biosynthesis* / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D008024:Ligands; D018102:Receptors, Leukotriene B4; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin; D007930:Leucine