Critical Role for Polar Residues in Coupling Leukotriene B4 Binding to Signal Transduction in BLT1

Abstract

Seven transmembrane receptors widely known as G-proteincoupled receptors (GPCRs) 4 (1, 2) mediate an array of physiological processes in response to such diverse agonists as peptides, amino acid derivatives, and lipids. Despite the great diversity in their ligands, the conserved motifs found across this superfamily and the limited interacting partners such as G-proteins (3) and ␤-arrestins (4) at the cytoplasmic interface point toward a common activation mechanism for GPCRs. GPCRs constitute the single largest group of molecules for drug targets due to their critical importance in mediating biological responses as well as their easy accessibility on the cell surface. However, very little structural information is available for GPCRs due to difficulties in purifying and obtaining crystal structures for this class of receptors. The availability of the rhodopsin crystal structure (5) combined with the approach of computational modeling and validation by site-directed mutagenesis has led to delineation of ligand-receptor interactions in a few GPCRs (6 -9). Some elements of the activation mechanism have been identified for individual GPCRs (1,6,8,[10][11][12]. Several studies employing site-directed mutagenesis have helped uncover critical interactions between residues in transmembrane domains of the GPCRs (reviewed in Ref. 1). The approach of computational modeling with validation by site-directed mutagenesis has led to significant increases in the understanding of the processes involved in GPCR activation (6 -9).Leukotriene B 4 (LTB 4 ) is a potent leukocyte chemoattractant and mediates its biological effects through two distinct GPCRs, the high affinity receptor BLT1 and the low affinity receptor BLT2 (13,14). Several recent studies suggested a direct and critical role for BLT1 in diverse inflammatory diseases such as arthritis (15, 16), atherosclerosis (17, 18), and asthma (19). Recently, the high affinity LTB 4 receptor, BLT1, was expressed in Escherichia coli and shown to form a functional pentameric complex with heterotrimeric G-proteins (20). Computational modeling has been used to investigate the potential role of the eighth helix in signaling of BLT1 (21,22). In addition, a recent study reported an LTB 4 binding site in BLT1 deduced from computational models (23). However, the exact nature of the LTB 4 binding site and the potential changes in receptor conformation following LTB 4 binding remain unknown.In this study, computational modeling together with sitedirected mutagenesis led to precise mapping and validation of the LTB 4 binding site in BLT1. Mutation of each of the residues predicted to be in the putative binding site resulted in reduced binding affinity. Furthermore, analysis of dynamic structures of the ligand-free and ligand-bound BLT1 allowed prediction of critical movements of transmembrane helices and essential

Phenylalanine 169 in the Second Extracellular Loop of the Human Histamine H4 Receptor Is Responsible for the Difference in Agonist Binding between Human and Mouse H4 Receptors

“…The EL2 has been suggested to be involved in the binding of diverse types of ligands, such as aminergic receptor ligands (Laurila et al, 2007;Scarselli et al, 2007), nicotinic acid (Tunaru et al, 2005), leukotriene B 4 (Basu et al, 2007), or vasopressin (Conner et al, 2007). The involvement of EL2 in the binding pocket of aminergic GPCRs has originally been suggested for the dopamine D 2 receptor on the basis of detailed substituted-cysteine accessibility analysis (Shi and Javitch, 2004).…”

Differential Signaling Defects Associated with the M201V Polymorphism in the Cysteinyl Leukotriene Type 2 Receptor

“…The binding site of cysteinyl leukotrienes to either of their receptors has not been determined. However, it has been shown that LTB 4 , a leukotriene lacking the cysteinyl moiety, uses, when binding to its high-affinity receptor (BLT1), residues in the C-terminal part of the ECL2 and TM5, regions possibly affected by the M201V mutation (Gearing et al, 2003;Sabirsh et al, 2006;Basu et al, 2007).…”

Structure-based design of inhibitors of CXCR4.

“…Mutations of the TM1 residue Asn36, TM3 residue Asp64, and the TM7 residues Ser277, Ser278, Ser279 and Asn281 produced a significant reduction in L TB4 mediated chemotaxis and calcium mobilization. Specifically, mutation of these residues prevented interactions between Asp64 and Asn281 as well as Asp64 and Asn36, which were required for transition of the molecule to its active state [151]. Similar studies involving the thyrotropin receptor [340] and histamine H1 receptor [341] revealed that mutation of TM regions could prevent transition of the receptors to the active state.…”

“…Some of the most common methods include assessment of CXCL 12-induced calcium mobilization [149][150][151], phosphorylation of secondary Signaling molecules such as ERK1/2, JNK, MAPK, and GSK 3a/~ [151][152][153], and internalization assays to assess the activity of ~-arrestin [142][143][144]. Assessment of CXCL 12-induced calcium mobilization involves pretreating cells with Fura-2 or Indo-I, fluorescent dyes which alter the ratio of their emissions in response to exposure to calcium.…”

“…Chemotaxis analysis for suspension 300.19 cells expressing hCXCR4-GFP or BL T1 was performed as previously described in Chapter 2 [151,281] .…”