Role of cholesterol-mediated effects in GPCR heterodimers
Introduction
The G protein-coupled receptor (GPCR) superfamily is the largest and a diverse group of transmembrane proteins that regulate several physiological processes (Pierce et al., 2002; Rosenbaum et al., 2009; Chattopadhyay, 2014). GPCRs are involved in signal transduction across the membrane in response to a diverse array of stimuli. Their central role in cell signaling makes them a crucial target for drug research, and ∼50 % of all currently marketed clinical drugs target GPCRs (Lagerström and Schiöth, 2008; Tautermann, 2014; Kumari et al., 2015). GPCRs consist of seven transmembrane helices and are interconnected by extracellular and intracellular loops (Katritch et al., 2012; Venkatakrishnan et al., 2013). As a consequence of such a molecular architecture, GPCR function is critically modulated by their membrane environment, mediated by membrane lipids and proteins (Chattopadhyay, 2014; Chakraborty and Chattopadhyay, 2015). For example, crystallography (Gimpl, 2016) and simulation studies (Sengupta and Chattopadhyay, 2015; Sengupta et al., 2018) of several GPCRs, such as the adenosine2A (A2A) (Lee and Lyman, 2012; Liu et al., 2012; Rouviere et al., 2017), serotonin1A (Sengupta and Chattopadhyay, 2012; Patra et al., 2015; Prasanna et al., 2016a) and β2-adrenergic receptor (Cherezov et al., 2007; Hanson et al., 2008; Cang et al., 2013; Prasanna et al., 2014) have reported multiple cholesterol interaction sites in the transmembrane domains. Importantly, several biochemical, biophysical and functional studies have suggested that GPCRs associate to form dimers or higher-order oligomers under physiological conditions (Jordan and Devi, 1999; Szidonya et al., 2008; Ferré et al., 2014; Franco et al., 2016). These associations occur between same (homodimer) or different (heterodimer) receptors. Formation of these complexes represents an additional mechanism for functional regulation of GPCRs (Jordan and Devi, 1999; Milligan, 2010; Ferré et al., 2014; Ferré, 2015).
Physiologically relevant heterodimers have been reported between specific subtypes of adenosine and dopamine receptors (Fuxe et al., 2007; Cabello et al., 2009; Trifilieff et al., 2011). Among these, the heterodimer complex of A2A and dopamine D2 (D2) receptors has been well characterized. In this complex, A2A receptor modulates ligand binding affinity, G-protein coupling and signaling of D2 receptor (Fuxe et al., 2007). As dopamine receptors are target for neuropsychiatric disorders such as Parkinson's disease and schizophrenia (Olanow et al., 2006; Seeman, 2013), it has been suggested that A2A receptor antagonists and agonists could be used as alternative targets (Fuxe et al., 2005, 2007). In fact, istradefylline, a selective A2A receptor antagonist has been approved for anti-Parkinson’s therapy (Dungo and Deeks, 2013; Pinna, 2014; Mori et al., 2015). The dopamine D3 (D3) receptor shows high degree of sequence homology with D2 receptor and is co-distributed in various regions of the ventral striatum (Volkow et al., 2015). The D3 receptor exhibits a lower expression and diversity in tissue distribution in comparison to the D2 receptor, but is more sensitive to changes in ligand concentration (its affinity for dopamine is 420-fold higher than that of D2 receptor) (Maramai et al., 2016). Antagonists specific to the D3 receptor have been suggested for treatment of psychotic and cognitive symptoms of schizophrenia (Gross et al., 2013; Sokoloff et al., 2013) and drug addiction (Heidbreder and Newman, 2010). Previous reports have shown that the A2A receptor modulates the ligand binding affinity of the D3 receptor (Hillefors et al., 1999). In addition, fluorescence resonance energy transfer (FRET) studies demonstrated that A2A receptor forms heteromeric complex with D3 receptor in which it antagonistically modulates signaling of D3 receptor (Torvinen et al., 2005). Although the D3 receptor appears to be less characterized in terms of its hetero-dimerization relative to the D2 receptor, its monomer structure has been crystallographically resolved (Chien et al., 2010). Understanding the heterodimer conformations of the A2A-D3 receptor could therefore provide important insight into the hetero-dimerization of these receptor families.
Membrane lipids have been shown to be important determinants of GPCR organization. Previous studies pointed toward modulation of higher-order oligomerization of the serotonin1A receptor (Pucadyil et al., 2005; Müller et al., 2007) by membrane cholesterol (Ganguly et al., 2011; Paila et al., 2011). More recently, quantitative analysis using photobleaching image correlation analysis (pbICS) showed a unique dimer-trimer equilibrium modulated by cholesterol (Chakraborty et al., 2018). Simulation studies have suggested that the presence of cholesterol induces increase in conformational plasticity of the serotonin1A receptor homodimer (Prasanna et al., 2016a). Similarly, specific interaction of lipids and cholesterol with GPCRs has been shown to modulate dimer conformation in the β2-adrenergic receptor (Cherezov et al., 2007; Prasanna et al., 2015) and the CXCR4 chemokine receptor (Pluhackova et al., 2016). A recent study on hetero-oligomerization of A2A and D2 receptors using bioluminescence resonance energy transfer (BRET) and coarse-grain simulations suggested that membranes containing docosahexanoic acid may enhance the rate of receptor oligomerization (Guixà-González et al., 2016). The presence of multiple cholesterol sites on the A2A receptor (Lee and Lyman, 2012; Liu et al., 2012; Rouviere et al., 2017) suggests a possible role of cholesterol in its constituent dimers, although this has not been explored.
In this work, we have examined the effect of membrane cholesterol on heterodimers of A2A and D3 receptor using multiple μs timescale coarse-grain molecular dynamics simulations. Analysis of simulation results reveal cholesterol interaction sites on A2A receptor surface, in agreement with crystal structure and atomistic simulations (Lee and Lyman, 2012; Liu et al., 2012). Interestingly, our results predict novel cholesterol binding sites on the D3 receptor. Our results suggest a large number of dimer conformations that are modulated by cholesterol interaction sites and local membrane perturbations, and highlight an important role of membrane cholesterol in modulating GPCR heterodimers. More importantly, these results would help in understanding the functional crosstalk between adenosine and dopamine receptors, with implications in designing therapeutic interventions in treatment of neuropsychiatric conditions.
Section snippets
System setup and simulation parameters
Structural models of the human A2A and D3 receptors were generated using the respective crystal structures (PDB ID: 3 EM L (Jaakola et al., 2008) and 3PBL (Chien et al., 2010)). The receptor structures were energy minimized and mapped to their coarse-grain representations. For monomer simulations, a single copy of the A2A receptor (amino acid residues 3-316) or the D3 receptor (amino acid residues 32-400) was inserted in pre-equilibrated bilayers. Two bilayers were considered: POPC bilayer and
Results
To analyze the cholesterol distribution around the receptors, we performed coarse-grain molecular dynamics simulations of A2A and D3 receptors, independently embedded in bilayers of POPC/30% cholesterol. In addition, a series of simulations with both A2A and D3 receptors were performed to investigate heterodimer formation in these receptors. Two membrane compositions were considered: POPC or POPC/30% cholesterol. A schematic representation of the initial conformation with the two
Discussion
Although the interaction of GPCRs at varying spatiotemporal scales (Sengupta et al., 2016) has now been established, understanding its functional consequences continues to be a challenging aspect (Chakraborty and Chattopadhyay, 2015; Sengupta et al., 2017). In a recent paper, a dynamic tunable equilibrium has been suggested and the conformation of the receptor dimers and higher oligomers may show considerable variations (Dijkman et al., 2018). In case of homodimers, GPCR association has been
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the Science and Engineering Research Board (Govt. of India) project (EMR/2016/002294) to D.S. and A.C. A.C. gratefully acknowledges support from SERB Distinguished Fellowship (Department of Science and Technology, Govt. of India). M.M. thanks the Department of Biotechnology (DBT-BINC), Govt. of India for the award of a Senior Research Fellowship. A.C. is a Distinguished Visiting Professor at Indian Institute of Technology (Bombay), and Adjunct Professor at Tata
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