Third Extracellular Loop (EC3)-N Terminus Interaction Is Important for Seven-transmembrane Domain Receptor Function

Abstract

G protein-coupled receptors (GPCRs), 2 often referred to as seven-transmembrane domain receptors, are one of the most biologically important superfamilies of receptors (1). Phylogenetic analysis classifies the superfamily of receptors into glutamate, rhodopsin, adhesive, frizzled, and secretin families (2).The rhodopsin family alone includes ϳ750 GPCRs (3). ϳ60% of GPCRs respond to sensory and olfactory neurons; ϳ30% respond to protein and peptide hormones, amino acids, biogenic amines, and lipids, whereas the remaining GPCRs respond to nutrients and metabolites. In response to ligands, the receptors recruit either G proteins or other intracellular effector proteins (4), which transduce the extracellular chemical signals and further stimulate intracellular signaling cascades (5). This extraordinary ability to sense such a diverse array of chemical signals with selective precision classifies GPCRs as a "druggable proteome" (6) that generates ϳ10% of the global pharma revenue (7).Given their cellular and pharmacological importance, understanding the receptor function at the molecular level is a prerequisite, and indeed the recent advancements in the high resolution crystals of rhodopsin-like GPCRs have been significantly informative (8 -12). Nevertheless, a central question remains how the canonical receptor topology interconverts between several possible conformations in response to diverse chemical stimuli. Traditionally, the GPCR activation mechanism is described as "molecular switches" identified in conserved "microdomains" (13) and recently reviewed elsewhere (14,15). Briefly, ligand/agonist binding or an activating point mutation (CAM) triggers a series of molecular macroswitches, such as the global rotamer toggling (16, 17) on TM6 or the disruption of an ionic lock (18,19), between TM3 and TM6, to unlock the G protein-binding site in the intracellular face of the receptors leading to G protein activation. However, ϳ80% of the rhodopsin family receptors do not have the putative residues to support the described "rotamer toggle" switch, the "ionic lock," or both (20). Although a universal activation mechanism is also suggested by the ability of human receptors to activate evolutionarily distant yeast G proteins, how this is accomplished is difficult to reconcile within a framework of low sequence homology (Յ20%) between receptors (21), the variable length of the peptide loops, and the diversity of N termini in receptors. Indeed, the significant variation observed in pharmacological properties of receptors (22,23) within the same family (24) does not favor a single, unified activation mechanism model.Within the rhodopsin family of GPCRs, the EC3 loop varies in length between 4 and 27 residues and plays a key role in the activation of the neuropeptide class of receptors (25). Minor alterations affecting its folding or structure have been shown to impair both ligand binding and signaling (26 -32). This study * This work was supported, in whole or in part, by National Institutes of Health Grants GM071634 and GM07...

Mutational Analysis of Atypical Chemokine Receptor 3 (ACKR3/CXCR7) Interaction with Its Chemokine Ligands CXCL11 and CXCL12

“…Nevertheless, the role of ECL4 for CXCL12 binding appears similar between CXCR4 (8,19,20) and ACKR3. Of note, ECL4 is also required for ligand-independent CXCR4 activation (19); whether this is also the case for ACKR3 remains to be studied.…”

“…This ECL4-forming disulfide bridge is essential for CXCR4 ligand binding and activation (19,20). ACKR3 has three cysteine residues in its N terminus, Cys-21, Cys-26, and Cys-34; based on sequence alignments, either Cys-21 or Cys-34 has been predicted to form ECL4 with Cys-287 7.25 in ECL3 (18,21) (residue numbering follows the Ballesteros-Weinstein scheme; Ref.…”

New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model

“…2A, B) [83–86]. Similar pharmacological and structural studies expanded these principles to other chemokine-CKR pairs, including CCR1 [81, 87, 88], CCR2 [18, 89, 90], CCR3 [91, 92], CCR5 [18, 88, 93–96], CXCR1 [97–99], CXCR2 [97, 98], CXCR3 [100], CXCR4 [82, 101], CX3CR1 [102]. …”

“…In addition to ECL2, ECLs 3 and 4 have been suggested to act as a tandem molecular switch required for CXCR4 activation [80, 101]. Using mutagenesis and a yeast-based Gα i protein activation screen, Rana and colleagues showed that interaction between TM7 and the CXCR4 N-terminus is essential for receptor activation, and that replacement of the disulfide-bonded cysteines with an electrostatic pair (Arg-Glu) conserves CXCR4 signaling.…”

Allosteric Modulation of a Cannabinoid G Protein-coupled Receptor

“…For example, mutation studies of the CXC chemokine receptor type 4 (i.e. CXCR4) suggest that an interaction between the EC-3 loop and the N terminus may form an "activation microswitch"; additionally, these results suggest that the conformation of the EC-3 loop may influence both basal and agonist-induced G protein-mediated signaling (53). In addition, mutation studies of the ␦-opioid receptor suggest that its EC-3 loop may form a tight hairpin structure and that the disruption of this conformation may trigger receptor activation (54).…”