Denis J. Dupré

The Role of Gβγ Subunits in the Organization, Assembly, and Function of GPCR Signaling Complexes

The role of Gbetagamma subunits in cellular signaling has become well established in the past 20 years. Not only do they regulate effectors once thought to be the sole targets of Galpha subunits, but it has become clear that they also have a unique set of binding partners and regulate signaling pathways that are not always localized to the plasma membrane. However, this may be only the beginning of the story. Gbetagamma subunits interact with G protein-coupled receptors, Galpha subunits, and several different effector molecules during assembly and trafficking of receptor-based signaling complexes and not simply in response to ligand stimulation at sites of receptor cellular activity. Gbetagamma assembly itself seems to be tightly regulated via the action of molecular chaperones and in turn may serve a similar role in the assembly of specific signaling complexes. We propose that specific Gbetagamma subunits have a broader role in controlling the architecture, assembly, and activity of cellular signaling pathways.

Seven Transmembrane Receptor Core Signaling Complexes Are Assembled Prior to Plasma Membrane Trafficking

Much is known about β2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the β2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-Gβγ complexes initially associate in the endoplasmic reticulum, whereas Gα subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.

Dopamine Receptor-interacting Protein 78 Acts as a Molecular Chaperone for Gγ Subunits before Assembly with Gβ

Heterotrimeric G proteins play a central role in intracellular communication mediated by extracellular signals, and both Gα and Gβγ subunits regulate effectors downstream of activated receptors. The particular constituents of the G protein heterotrimer affect both specificity and efficiency of signal transduction. However, little is known about mechanistic aspects of G protein assembly in the cell that would certainly contribute to formation of heterotrimers of specific composition. It was recently shown that phosducin-like protein (PhLP) modulated both Gβγ expression and subsequent signaling by chaperoning nascent Gβ and facilitating heterodimer formation with Gγ subunits (Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E., and Willardson, B. M. (2005) EMBO J. 24, 1965-1975; Humrich, J., Bermel, C., Bunemann, M., Harmark, L., Frost, R., Quitterer, U., and Lohse, M. J. (2005) J. Biol. Chem. 280, 20042-20050). Here we demonstrate using a variety of techniques that DRiP78, an endoplasmic reticulum resident protein known to regulate the trafficking of several seven transmembrane receptors, interacts specifically with the Gγ subunit but not Gβ or Gα subunits. Furthermore, we demonstrate that DRiP78 and the Gβ subunit can compete for the Gγ subunit. DRiP78 also protects Gγ from degradation until a stable partner such as Gβ is provided. Furthermore, DRiP78 interaction may represent a mechanism for assembly of specific Gβγ heterodimers, as selectivity was observed among Gγ isoforms for interaction with DRiP78 depending on the presence of particular Gβ subunits. Interestingly, we could detect an interaction between DRiP78 and PhLP, suggesting a role of DRiP78 in the assembly of Gβγ by linking Gγ to PhLP·Gβ complexes. Our results, therefore, suggest a role of DRiP78 as a chaperone in the assembly of Gβγ subunits of the G protein.

Type 1 cannabinoid receptor ligands display functional selectivity in a cell culture model of striatal medium spiny projection neurons.

Background: To understand the differential response to cannabinoids, we examined the functional selectivity of type 1 cannabinoid receptor (CB1) agonists in a cell model of striatal neurons. Results: 2-Arachidonylglycerol, Δ9-tetrahydrocannabinol, and CP55,940 were arrestin2-selective; endocannabinoids and WIN55,212-2 activated Gαi/o, Gβγ, and Gαq; and cannabidiol activated Gαs independent of CB1. Conclusion: Cannabinoids displayed functional selectivity. Significance: CB1 functional selectivity may be exploited to maximize therapeutic efficacy. Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC)) on arrestin2-, Gαi/o-, Gβγ-, Gαs-, and Gαq-mediated intracellular signaling in the mouse STHdhQ7/Q7 cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gαi/o and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gαq-dependent pathways. CP55,940 and CBD both signaled through Gαs. CP55,940, but not CBD, activated downstream Gαs pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.

The apelin receptor: Physiology, pathology, cell signalling, and ligand modulation of a peptide-activated class A GPCR

The apelin receptor (AR or APJ) is a class A (rhodopsin-like) G-protein-coupled receptor with wide distribution throughout the human body. Activation of the AR by its cognate peptide ligand, apelin, induces diverse physiological effects including vasoconstriction and dilation, strengthening of heart muscle contractility, angiogenesis, and regulation of energy metabolism and fluid homeostasis. Recently, another endogenous peptidic activator of the AR, Toddler/ELABELA, was identified as having a crucial role in zebrafish (Danio rerio) embryonic development. The AR is also implicated in pathologies including cardiovascular disease, diabetes, obesity, and cancer, making it a promising therapeutic target. Despite its established importance, the precise roles of AR signalling remain poorly understood. Moreover, little is known about the mechanisms of peptide-AR activation. Additional complexity arises from modulation of the AR by 2 endogenous peptide ligands, both with multiple bioactive isoforms of variable length and distribution. The various apelin and Toddler/ELABELA isoforms may also produce distinct cellular effects. Further complexity arises through formation of functionally distinct heterodimers between the AR and other G-protein-coupled receptors. This minireview outlines key (patho)physiological actions of the AR, addresses what is known about signal transduction downstream of AR activation, and concludes by discussing unique properties of the endogenous peptidic ligands of the AR.

Na+/H+ Exchanger Regulatory Factor-1 Is Involved in Chemokine Receptor Homodimer CCR5 Internalization and Signal Transduction but Does Not Affect CXCR4 Homodimer or CXCR4-CCR5 Heterodimer

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 is also the principal co-receptor for macrophage-tropic strains of human immunodeficiency virus, type 1 (HIV-1), and efforts have been made to develop ligands to inhibit HIV-1 infection by promoting CCR5 receptor endocytosis. Given the nature of GPCRs and their propensity to form oligomers, one can consider ligand-based therapies as unselective in terms of the oligomeric composition of complexes. For example, a ligand targeting a CCR5 homomer could likely induce signal transduction on a heteromeric CCR5-CXCR4. Other avenues could therefore be explored. We identified a receptor adaptor interacting specifically with one receptor complex but not others. NHERF1, an adaptor known for its role in desensitization, internalization, and regulation of the ERK signaling cascade for several GPCRs, interacts via its PDZ2 domain with the CCR5 homodimer but not with the CXCR4-CCR5 heterodimer or CXCR4 homodimer. To further characterize this interaction, we also show that NHERF1 increases the CCR5 recruitment of arrestin2 following stimulation. NHERF1 is also involved in CCR5 internalization, as we demonstrate that co-expression of constructs bearing the PDZ2 domain can block CCR5 internalization. We also show that NHERF1 potentiates RANTES (regulated on activation normal T cell expressed and secreted)-induced ERK1/2 phosphorylation via CCR5 activation and that this activation requires NHERF1 but not arrestin2. Taken together, our results suggest that oligomeric receptor complexes can associate specifically with partners and that in this case NHERF1 could represent an interesting new target for the regulation of CCR5 internalization and potentially HIV infection.

Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking.

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55(1.59). Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N46(1.50) is central to a disruption in helical character. G42(1.46), G45(1.49) and N46(1.50), which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

Anterograde Trafficking of CXCR4 and CCR2 Receptors in a Prostate Cancer Cell Line

Background: Most prostate cancer-related deaths result from metastasis. CXCR4 and CCR2 are known to govern cellular processes resulting in cell migration, proliferation and survival. These receptors are expressed at low levels on normal prostate cells and are highly expressed on malignant and metastatic prostate cancer cells. Signaling of these receptors is relatively well understood, but processes governing their expression at the cell membrane are not. PC3 prostate cancer cells were used to demonstrate the importance of various Rab GTPases on cell surface expression and signaling of CXCR4 and CCR2, along with the CXCR4/CCR2 heterodimer. Methods: PC3 prostate cancer cells were transfected with select Rab GTPase wild-type and dominant negative constructs. Effects of each Rab GTPase on endogenous cell surface expression of the individual receptors, along with the overexpressed CXCR4/CCR2 heterodimer, were determined by biotin-streptavidin cell surface assays. These results were corroborated by assessing signal transduction, measured by focal adhesion kinase (FAK) activation. Conclusion: Rab GTPases required for cell surface expression and signal transduction of CXCR4 or CCR2 differ from those required for the CXCR4/CCR2 heterodimer. Determining trafficking regulators of two key receptors involved in the metastatic transition may identify new targets to restrict expression of chemokine receptors employed during metastasis.

Dopamine Receptor-Interacting Protein 78 Acts as a Molecular Chaperone for CCR5 Chemokine Receptor Signaling Complex Organization

Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 and CXCR4 act as co-receptors for human immunodeficiency virus (HIV) and several efforts have been made to develop ligands to inhibit HIV infection by blocking those receptors. Removal of chemokine receptors from the cell surface using polymorphisms or other means confers some levels of immunity against HIV infection. Up to now, very limited success has been obtained using ligand therapies so we explored potential avenues to regulate chemokine receptor expression at the plasma membrane. We identified a molecular chaperone, DRiP78, that interacts with both CXCR4 and CCR5, but not the heterodimer formed by these receptors. We further characterized the effects of DRiP78 on CCR5 function. We show that the molecular chaperone inhibits CCR5 localization to the plasma membrane. We identified the interaction region on the receptor, the F(x)6LL motif, and show that upon mutation of this motif the chaperone cannot interact with the receptor. We also show that DRiP78 is involved in the assembly of CCR5 chemokine signaling complex as a homodimer, as well as with the Gαi protein. Finally, modulation of DRiP78 levels will affect receptor functions, such as cell migration in cells that endogenously express CCR5. Our results demonstrate that modulation of the functions of a chaperone can affect signal transduction at the cell surface.

Chaperones contribute to G protein coupled receptor oligomerization, but do not participate in assembly of the G protein with the receptor signaling complex

Background Previous studies have demonstrated that seven transmembrane receptors (7TM-Rs) can associate with various chaperones to control their maturation and export. It has been shown for a few years now that 7TM-Rs can form homo or heterooligomeric complexes. Due to the difficulty to study heterooligomers in a context devoid of homooligomers signaling, very little is known on heterooligomerization. β2AR-AT1R receptor complexes have been found on cells and ligand activation of one receptor affects signaling of the partner. Yet, very little is known about the mechanisms linking those receptors together. We propose to examine the role of chaperones in the maturation of homo- and heterodimers of the β2AR and AT1R. It would not be surprising that strict cellular mechanisms exist to ensure that only properly folded receptors are inserted into the plasma membrane. Results Our goal is to understand the process whereby the adrenergic and angiotensin receptors attain their proper mature conformation. We determined whether any of the common chaperones are physically associated with the fully and/or immature β2AR and AT1R receptors forms and if they play any role in the selective recruitment of G proteins subunits to receptor complexes. Our results suggest that when a pair of receptors is expressed in such way that one is retained in the endoplasmic reticulum (ER), this immature receptor will dictate the chaperones interacting with the receptor complex. We showed that ERp57 is important for receptor dimerization of AT1R homo and β2AR/AT1R receptor dimers, but plays no role in the β2AR homodimerization. Then, we verified if some of those chaperones could play a role in the assembly of the heterotrimeric G protein subunits with the receptor complex, but none appeared to be essential. Conclusions Overall, our results suggest that variations among receptor oligomers occur early in the synthesis/maturation processes, and that chaperones will interact more specifically with some receptor pairs than others to allow the formation of certain receptor pairs, while others will contribute to the folding and maturation of receptors without any effect on receptor assembly within a signaling complex.