Probing the molecular mechanisms of GPCR functional selectivity in live cells

A fundamental unanswered question in GPCR signaling is the role of structural conformations of the GPCR in G protein selection. An emerging view from several studies is that GPCRs are not simple ‘on-off’ switches, but adopt multiple conformations in a ligand-dependent manner. In turn, these ligands elicit diverse functional responses through the activation of distinct G protein heterotrimers or G protein independent effectors such as arrestins. The conformational plasticity of GPCRs addresses the phenomenon of functional selectivity, wherein the same GPCR can elicit diverse ligand-dependent responses. Currently there is no direct method to link ligand-specific GPCR conformations observed in structural studies to differential downstream responses. The laboratory has used protein engineering to develop a GPCR conformational biosensor (Malik et al., JBC 2013) that can detect G protein-specific conformations.  We have used this technology to demonstrate a Gi-specific conformation in the β2-adrenergic receptor induced by the β-blocker metoprolol (Fig. 1). Currently several laboratories are using our sensors to profile the functional selectivity of a range of GPCRs.

We have also used protein engineering to map the rules of the GPCR-G protein interaction. This study has yielded surprising insights into the affinities of GPCRs for G proteins, and the functional consequences of these interaction profiles in downstream responses. Our findings provide a strong foundation for GPCR-G protein pairing, and in due process have yielded a simple yet powerful genetically encoded technique to ‘tune’ G protein selection in live cells. Current efforts are focused on using this technique to (a) link G protein selection to physiological responses in cells and model organisms; and (b) define a quantitative ‘bias factor’ for ligands that is independent of the cell type being examined.

Selected Publications

  • Structural elements in the Gαs and Gαq C-termini that mediate selective GPCR signaling. A. Semack*, M. Sandhu*, R. U. Malik, N. Vaidehi and S. Sivaramakrishnan Journal of Biological Chemistry Jun 22, 2016 [Epub date] *Equal contribution 
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  • Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel FRET Sensor. M. Ritt and S. Sivaramakrishnan. Molecular Pharamcology Jan 22, 2016 [Epub date] 
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  • Dynamic coupling and allosteric networks in the alpha subunit of heterotrimeric G proteins. X.Q. Yao*, R.U. Malik*, N.W. Griggs, L. Skjaerven, J.R. Traynor, S. Sivaramakrishnan, B.J. Grant. Journal of Biological Chemistry Dec 24, 2015 [Epub date] *Equal contribution. 
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  • Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells. R.U. Malik , M. Ritt, B.T. Devree, R.R. Neubig, R.K. Sunahara, S. Sivaramakrishnan. Journal of Biological Chemistry 2013, 288(24), 17167-78. 
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Figure 1: Distinct structural mechanisms of β2-AR agonists and inverse agonists. In the absence of ligand (basal state), only a small proportion of the β2-AR population adopts Gs conformations. Isoproterenol (agonist) treatment destabilizes the DRY ionic-lock and enhances interaction with the Gαs c-terminus, resulting in activation of adenylyl cyclase (AC). Conversely, ICI118,551 (inverse agonist), reinforces the DRY ionic-lock, and shifts the equilibrium toward inactive conformations. Biased agonist (metoprolol) stabilizes Gi conformations, promoting Gi dependent inhibition of AC (Malik et al., JBC, 2013).