Cholesterol Is a Dose-Dependent Positive Allosteric Modulator of CCR3 Ligand Affinity and G Protein Coupling

Abstract

Cholesterol as an allosteric modulator of G protein-coupled receptor (GPCR) function is well documented. This quintessential mammalian lipid facilitates receptor–ligand interactions and multimerization states. Functionally, this introduces a complicated mechanism for the homeostatic modulation of GPCR signaling. Chemokine receptors are Class A GPCRs responsible for immune cell trafficking through the binding of endogenous peptide ligands. CCR3 is a CC motif chemokine receptor expressed by eosinophils and basophils. It traffics these cells by transducing the signal stimulated by the CC motif chemokine primary messengers 11, 24, and 26. These behaviors are close to the human immunoresponse. Thus, CCR3 is implicated in cancer metastasis and inflammatory conditions. However, there is a paucity of experimental evidence linking the functional states of CCR3 to the molecular mechanisms of cholesterol–receptor cooperativity. In this vein, we present a means to combine codon harmonization and a maltose-binding protein fusion tag to produce CCR3 from E. coli. This technique yields ∼2.6 mg of functional GPCR per liter of minimal media. We leveraged this protein production capability to investigate the effects of cholesterol on CCR3 function in vitro. We found that affinity for the endogenous ligand CCL11 increases in a dose-dependent manner with cholesterol concentration in both styrene:maleic acid lipid particles (SMALPs) and proteoliposomes. This heightened receptor activation directly translates to increased signal transduction as measured by the GTPase activity of the bound G-protein α inhibitory subunit 3 (Gαi3). This work represents a critical step forward in understanding the role of cholesterol-GPCR allostery in regulation of signal transduction.

Comparative Study of State-Dependent Cholesterol Binding Sites in Adenosine A2A and A1 Receptors Using Coarse-Grained Molecular Dynamics Simulations in Biologically Relevant Membranes

“…Thus, the binding affinity of a selective antagonist was enhanced by cholesterol depletion in the case of A2AR selective antagonist 59 and was reduced in presence of cholesterol for chemokine receptor type 5 (CCR5). 123 In contrast, the binding affinity of an agonist was increased in a dose-dependent manner with cholesterol concentration for the chemokine receptor CCR3 124 or oxytocin receptor (OTR) 124 125 and was reduced by cholesterol depletion in the case of 5-HT1A receptors. 116 The first structural evidence for site-specific cholesterol binding in GPCRs was provided by early X-ray structures of inactive states of β2AR 25 and A2AR, 10 in which cholesterol binds in distinct TM positions forming cavities.…”

“…It has also been suggested that cholesterol binding to certain cavities can stabilize the active or inactive state and can shift the equilibrium accordingly. For example, the binding affinity of an agonist has been shown to increase in a dose-dependent manner with cholesterol concentration for the chemokine receptor CCR3 124 or oxytocin receptor (OTR) 124,125 or for an antagonist against angiotensin II receptor type 1, 137 and has been shown to reduce by cholesterol depletion in the case of 5-HT1A receptors. 116 Thus, these stable, long residence time cholesterol binding sites can be used for the design of allosteric modulators which can bind through lipid pathways as has been described for CB1 receptor 138,135 and can be accomplished for other GPCRs, e.g.…”

G-Protein-Coupled Receptors in Rheumatoid Arthritis: Recent Insights into Mechanisms and Functional Roles

“…CCR3 may be coupled with Gai/o (42). The ligands of CCR3 are CCL4, CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL15 (HCC-1), CXCL10 (interferon (IFN)-g inducible protein-10), CCL11 (eotaxin), CCL13, CCL24 (eotaxin-2), and CCL26 (eotaxin-3) (17,22,31).…”

A Cholesterol Dimer Stabilizes the Inactivated State of an Inward‐Rectifier Potassium Channel

“…[3] The functional interplay between cholesterol and bilayer components is biologically essential. [4] Cholesterol is facilitates G proteincoupled receptor (GPCR) dimerization [5] and activity, [6] promotes the open state of acetylcholine receptors, and attenuates ion channel activity. [7] Extant membrane protein structures contain cholesterol, including GPCRs, [8][9][10] ATP-binding cassettes, [11] Na + -K + -ATPases, [12,13] and Tetraspanin CD-81.…”