Interaction of G protein coupled receptors and cholesterol

Highlights

• Crystal structures of some GPCRs show cholesterol molecules at various positions.

• Different cholesterol binding sites in GPCRs are described.

• Cholesterol acts as stabilizer of receptor conformations.

Abstract

G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs.

Introduction

The human genome encodes ∼800 G protein coupled receptors (GPCRs) that form the largest receptor superfamily in eukaryotic cells. These receptors display a highly conserved seven transmembrane core architecture and are fundamentally important in the signal transduction and cellular response to different kinds of extracellular stimuli. They are classified into five main families: class A/rhodopsin, class B/secretin, class C/glutamate, class F/frizzled, and the adhesion family (Lagerstrom and Schioth, 2008), with class-A/rhodopsin GPCRs representing the majority of GPCRs (∼85% in human genome). Over the last years, enormous progress on receptor structures has been achieved. This was the result of a fruitfull combination of advances in receptor engineering (e.g. the introduction of truncations, conformational stabilization, chimeric constructs), development of new detergents, and improvements in the techniques of crystallization and crystallography (Piscitelli et al., 2015). Until now, crystal structures of all classes of GPCRs (except the adhesion family) have been reported (see gpcr.usc.edu or gpcrdb.org for details).

The canonical activation of GPCRs is a three-step process: (i) ligand binding to the receptor stabilizing an active receptor conformation, (ii) GDP dissociation from the Gα subunit (rate-limiting) and its exchange for GTP, and (iii) dissociation of the G protein heterotrimer into the two membrane-anchored subunits Gα-GTP and Gβγ. Signaling could not only occur either through classical G protein activation, but also through β-arrestin-mediated pathways, or through either of these pathways, termed biased signaling. GPCRs can adopt various active and inactive conformations that can be stabilized by appropriate ligands. In some cases, receptors are able to induce signaling even in the absence of any ligand. When inverse agonists bind to these constitutively active receptors, their basal activity can be reduced. Thus, GPCRs have to be regarded as highly dynamic, versatile signaling proteins rather than simple toggle switches (Nygaard et al., 2013).

All efforts to purify and crystallize GPCRs have to overcome the high intrinsic conformational flexibility and dynamics of these receptors within the membrane bilayer. So it is no surprise that almost all successfull structural determinations that have been achieved to date required receptor complexes with stabilizing ligands irrespective of their nature (agonists, antagonists, inverse agonists). Moreover, an active state ternary complex of β2AR with a G protein (Rasmussen et al., 2011) and with β-arrestin (Kang et al., 2015) were reported.

In view of the abundance of cholesterol in the plasma membrane of eukaryotic cells, all integral membrane proteins are constantly in close proximity to cholesterol molecules. This is particularly true for the GPCRs. Owing to their seven transmembrane helices large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. It is difficult to clarify whether such cholesterol dependence is based on direct interaction with cholesterol or on indirect effects caused by the influence of cholesterol on the biophysical state of the membrane. Some GPCRs have been shown to be functionally dependent on the specific presence of cholesterol (Burger et al., 2000, Pucadyil and Chattopadhyay, 2006, Paila and Chattopadhyay, 2010). Methods and approaches to study the cholesterol binding have been recently reviewed (Gimpl, 2010).