Regulation of G protein-coupled receptors by palmitoylation and cholesterol

Due to their membrane location, G protein-coupled receptors (GPCRs) are subject to regulation by soluble and integral membrane proteins as well as membrane components, including lipids and sterols. GPCRs also undergo a variety of post-translational modifications, including palmitoylation. A recent article by Zheng et al. in BMC Cell Biology demonstrates cooperative roles for receptor palmitoylation and cholesterol binding in GPCR dimerization and G protein coupling, underlining the complex regulation of these receptors

Approaches to Characterize and Quantify Oligomerization of GPCRs

Fluorescence resonance energy transfer (FRET) is an approach widely used to detect protein–protein interactions in live cells. This approach is based on the sensitization of an “acceptor” molecule by the energy transfer from a “donor” when there is an overlap between the emission spectrum of the “donor” and the excitation spectrum of the “acceptor” and close proximity between the two fluorophore species (in the region of 8 nm). Various methods exist to quantify FRET signals: here, we describe the application of homogeneous time-resolved FRET (htrFRET) combined with Tag-lite™ technology and its application to determine not only protein–protein interactions but also the capability of GPCR mutant variants to form homomers compared to the wild type GPCR within the plasma membrane of transfected cells

Internalization Dissociates β2-Adrenergic Receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β2-adrenergic receptors (β2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between β2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β2ARs and endosome markers increases. Energy transfer between β2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β2ARs associate transiently with each other in the plasma membrane, or that β2AR dimers or oligomers are actively disrupted during internalization

Quaternary structure of a G-protein coupled receptor heterotetramer in complex with Gi and Gs

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function

Synergistic interplay of Gβγ and phosphatidylinositol 4,5-bisphosphate dictates Kv7.4 channel activity.

Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP2. In HEK cells stably expressing Kv7.4, PIP2 or Gβγ increased open probability in a concentration dependent manner. Depleting PIP2 prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP2-induced current enhancement. A combination of PIP2 and Gβγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP2 depletion or Gβγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gβγ

Regulation of GIP and GLP1 Receptor Cell Surface Expression by N-Glycosylation and Receptor Heteromerization

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer

Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

Applications of fluorescence and bioluminescence resonance energy transfer to drug discovery at G protein coupled receptors

The role of G protein coupled receptors (GPCRs) in numerous physiological processes that may be disrupted or modified in disease makes them key targets for the development of new therapeutic medicines. A wide variety of resonance energy transfer (RET) techniques such as fluorescence RET and bioluminescence RET have been developed in recent years to detect protein–protein interactions in living cells. Furthermore, these techniques are now being exploited to screen for novel compounds that activate or block GPCRs and to search for new, previously undiscovered signaling pathways activated by well-known pharmacologically classified drugs. The high resolution that can be achieved with these RET methods means that they are well suited to study both intramolecular conformational changes in response to ligand binding at the receptor level and intermolecular interactions involving protein translocation in subcellular compartments resulting from external stimuli. In this review we highlight the latest advances in these technologies to illustrate general principles