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 (Gα i3). This work represents a critical step forward in understanding the role of cholesterol-GPCR allostery in regulation of signal transduction.
Highlights
G protein-coupled receptors (GPCRs) are integral membrane proteins comprising a canonical seventransmembrane alpha helical architecture (Rosenbaum et al, 2009)
CCL11 and G-protein α inhibitory subunit 3 (Gαi3) were DUMB optimized as a matter in expression optimization, attaining ∼2.6 ± 0.3 and 15.1 ± 0.3 mg/L, respectively, for each, from M9 minimal media
The total yield of functional, folded protein is the main bottleneck in the study of GPCRs
Summary
G protein-coupled receptors (GPCRs) are integral membrane proteins comprising a canonical seventransmembrane alpha helical architecture (Rosenbaum et al, 2009). In response to external stimuli, this helical bundle undergoes a conformational change that is recognized by an intracellular heterotrimeric G protein (Kim et al, 2013). This molecular recognition event leads to an exchange of bound GDP for GTP in the G protein, triggering dissociation of the α and βc subunits (Figure 1) (Sullivan et al, 1987). The α subunit acts as an effector to influence downstream events such as modulation of adenylate cyclase functionality (Federman et al, 1992), while the βc subunit can trigger cleavage of phosphatidylinositol-(4,5)-bisphosphate (PIP2) (Katz et al, 1992) and ion channel activation (Pegan et al, 2005; Nishida et al, 2007).
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