Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach

<div><p>G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β₂-adrenergic receptor (β₂AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β₂AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β₂AR pull-down, 242 proteins in the inverse agonist-activated β₂AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β₂AR-interacting proteins isolated was confirmed by Western blot; three known β₂AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β₂AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β₂AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.</p> </div

Summary of selected identified proteins.

<p>Summary of selected identified proteins.</p

Cellular Functions of Identified Proteins.

<p>Normal: proteins identified both in BI-occupied and Cz-occupied β₂ARr•HDL pull-downs.</p><p>(<i>Italic</i>): proteins identified in BI-occupied β₂ARr•HDL pull-downs. (<b>Bold</b>): proteins identified in Cz-occupied β₂AR•rHDL pull-downs.</p

Co-immunoprecipitation of β₂AR-interacting proteins in the adult rat heart

<p>cytosol. A) Reconstitution process of β₂AR into HDL particles. B) Coomassie-stained SDS-PAGE gel of β₂AR•rHDL-interacting proteins. C) Coomassie-stained SDS-PAGE gel of empty HDL-interacting proteins. BI: 50 µM BI-167107, Cz: 50 µM Carazolol. Gel pieces (1A through 3H for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054942#pone-0054942-g001" target="_blank">Figure 1B, and 1A</a> through <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054942#pone-0054942-g002" target="_blank">2F</a> for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054942#pone-0054942-g001" target="_blank">Figure 1C</a>) were cut out for mass spectrometry-based protein identification.</p

Validation of identified proteins by Western blotting.

<p>β₂AR•rHDL-pull down samples were run on SDS-PAGE gel, transferred to PVDF membrane, and analyzed by Western blotting using antibodies specific for each binding partners. The figures are the representative of at least two independent experiments. BI: 50 µM BI-167107, Cz: 50 µM Carazolol.</p

The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the β2 adrenergic receptor (β2AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The β2AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the β2AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the β2AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the β2AR and the structural basis for ligand efficacy

Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity