Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M₂ muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[³H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M₂ receptor over monomers as the biologically relevant state for coupling to G proteins.     

Structures of Gα Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms

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Intracellular Transfer of Na+ in an Active-State G-Protein-Coupled Receptor

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G Protein-coupled Receptor (GPCR) Signaling via Heterotrimeric G Proteins from Endosomes

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a “second wave” of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors.     

Deorphanization of Dresden G Protein-Coupled Receptor for an Odorant Receptor

Dresden G protein-coupled receptor (D-GPCR) is one of orphan G protein-coupled receptors (GPCR). Here we report the identification of the ligands and the characterization of D-GPCR. We investigated over 5000 compounds to evoke the response mediated by D-GPCR and identified 3-methyl-valeric acid and 4-methyl-valeric acid as agonists using a cAMP assay. It is of interest that they dramatically enhanced the intracellular cAMP accumulation and the CRE-luciferase activity in CHO-K1 cells and HEK293 cells expressing the chimeric protein of D-GPCR with a rhodopsin-tag at its N-terminus. Our results established new characteristics of D-GPCR as an olfactory receptor. First, agonists of D-GPCR belong to odorants. Second, D-GPCR mRNA is expressed in the olfactory bulb. In addition, D-GPCR was reported to have similar sequences and its genome locus nearby other olfactory receptors. These results suggest D-GPCR is an olfactory receptor.     

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 ⁴²⁰AKKAA⁴²⁴ mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gα_q association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.     

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM₁ and AM₂ receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM₁ and AM₂ receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM₁ and AM₂ receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.     

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.     

Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles

The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for α-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [¹⁵N], [¹⁵N, ¹³C], [¹⁵N, ¹³C, ²H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three α-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg⁵⁸ site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 ± 0.10 Å, 0.40 ± 0.13 Å, and 0.57 ± 0.19 Å, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G⁵⁶VRSG⁶⁰ region. ¹⁵N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor.     

Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor

The G protein-coupled β₂-adrenoreceptor (β₂AR) signals through the heterotrimeric G proteins G_s and G_i and β-arrestin. As such, the energy landscape of β₂AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β₂AR with a trifluoromethyl probe, ¹⁹F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β₂AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β₂AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.     

Crystal Structure of CC Chemokine Receptor 2A in Complex with an Orthosteric Antagonist Provides Insights for the Design of Selective Antagonists

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Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology

Over the past half-century, The Journal of Biological Chemistry has been the venue for many landmark publications on the topic of G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors). The GPCR superfamily in humans is composed of about 800 members, and is the target of about one-third of all pharmaceuticals. Most of these drugs target a very small subset of GPCRs, and do so by mimicking or competing with endogenous hormones and neurotransmitters. This thematic minireview series examines some emerging trends in GPCR drug discovery. The first article describes efforts to systematically interrogate the human “GPCR-ome,” including more than 150 uncharacterized “orphan” receptors. The second article describes recent efforts to target alternative receptor binding sites with drugs that act as allosteric modulators of orthosteric ligands. The third article describes how the recent expansion of GPCR structures is providing new opportunities for computer-guided drug discovery. Collectively, these three articles provide a roadmap for the most important emerging trends in GPCR pharmacology.     

Detection of G Protein-selective G Protein-coupled Receptor (GPCR) Conformations in Live Cells

Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved (E/D)RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D)RY motif (ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the β2-AR (E/D)RY ionic lock enhances interaction with G_s. However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D)RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of β2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with G_s and promotes G_i binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.     

Crystal Structures of S120G Mutant and Wild Type of Human Nucleoside Diphosphate Kinase A in Complex with ADP

Nm23 was the first metastasis suppressor gene identified. This gene encodes a NDP kinase that also exhibits other properties like histidine protein kinase and interactions with proteins and DNA. The S120G mutant of NDPK-A has been identified in aggressive neuroblastomas and has been found to reduce the metastasis suppressor effect of Nm23. In order to understand the differences between the wild type and the S120G mutant, we have determined the structure of both mutant and wild type NDPK-A in complex with ADP. Our results reveal that there are no significant changes between the two enzyme versions even in the surroundings of the catalytic histidine that is required for NDP kinase activity. This suggests that the S120G mutation may affect an other protein property than NDP kinase activity.     

Human Substance P Receptor Expressed in Sf9 Cells Couples with Multiple Endogenous G Proteins

Abstract

To identify the G proteins involved in the function of human substance P receptor (hSPR), the receptor was expressed in Sf9 cells using the baculovirus expression system. Maximal hSPR expression was up to 65 pmol/mg membrane protein. The following data indicated that hSPR in Sf9 membranes is coupled to endogenous G proteins: 1) binding of agonist radioligand [¹²⁵I]BHSP to the receptor was sensitive to guanine nucleotides; and 2) stimulation of the receptor increased [³⁵S]GTPγS binding. The hSPR-associated G proteins were identified by photoaffinity labeling with [α-³²P]-azidoanilido GTP ([α-³²P]AAGTP), followed by immunoprecipitation of the labeled G proteins with antibodies specific for various Gα-subunits. These experiments showed that stimulation of hSPR in Sf9 membranes activated multiple endogenous G proteins including Gα_o, Gα^q/11, and Gα. While hSPR's ability to associate with G^q/11 is well-documented, the present study provides the first evidence of hSPR's potential to activate Gα_o and Gα_s.     

RACK1 Regulates the Cell Surface Expression of the G Protein-Coupled Receptor for Thromboxane A2

We used the yeast two-hybrid system to screen for proteins that interact with the C-terminus of the β isoform of the thromboxane A₂ receptor (TPβ). This screen identified receptor for activated C-kinase 1 (RACK1) as a new TPβ-interacting protein. Here, we show that RACK1 directly binds to the C-terminus and the first intracellular loop of TPβ. The TPβ–RACK1 association was further confirmed by co-immunoprecipitation studies in HEK293 cells and was not modulated by stimulation of the receptor. We observed that cell surface expression of TPβ was increased when RACK1 was overexpressed, while it was inhibited when endogenous RACK1 expression was knocked down by small interfering RNA. Confocal microscopy confirmed the impaired cell surface expression of TPβ and suggested that the receptors remained predominantly localized in the endoplasmic reticulum (ER) in RACK1-depleted cells. Confocal microscopy also revealed that a transient TPβ–RACK1 association takes place in the ER. The effect of RACK1 on receptor trafficking to the cell surface appears to be selective to some G protein-coupled receptors (GPCRs) because inhibition of RACK1 expression also affected cell surface targeting of the angiotensin II type 1 receptor and CXCR4 but not of β₂-adrenergic and prostanoid DP receptors. Our data demonstrate for the first time a direct interaction between RACK1 and a GPCR and identify a novel role for RACK1 in the regulation of the transport of a membrane receptor from the ER to the cell surface.     

A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, (−)-7′-isothiocyanato-11-hydroxy-1′,1′dimethylheptyl-hexahydrocannabinol (AM841), enters the cannabinoid CB2 receptor via the lipid bilayer (Pei, Y., Mercier, R. W., Anday, J. K., Thakur, G. A., Zvonok, A. M., Hurst, D., Reggio, P. H., Janero, D. R., and Makriyannis, A. (2008) Chem. Biol. 15, 1207–1219). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. Here, we test the hypothesis that the endogenous cannabinoid sn-2-arachidonoylglycerol (2-AG) attains access to the CB2 receptor via the lipid bilayer. To this end, we have employed microsecond time scale all-atom molecular dynamics (MD) simulations of the interaction of 2-AG with CB2 via a palmitoyl-oleoyl-phosphatidylcholine lipid bilayer. Results suggest the following: 1) 2-AG first partitions out of bulk lipid at the transmembrane α-helix (TMH) 6/7 interface; 2) 2-AG then enters the CB2 receptor binding pocket by passing between TMH6 and TMH7; 3) the entrance of the 2-AG headgroup into the CB2 binding pocket is sufficient to trigger breaking of the intracellular TMH3/6 ionic lock and the movement of the TMH6 intracellular end away from TMH3; and 4) subsequent to protonation at D3.49/D6.30, further 2-AG entry into the ligand binding pocket results in both a W6.48 toggle switch change and a large influx of water. To our knowledge, this is the first demonstration via unbiased molecular dynamics that a ligand can access the binding pocket of a class A G protein-coupled receptor via the lipid bilayer and the first demonstration via molecular dynamics of G protein-coupled receptor activation triggered by a ligand binding event.     

Calcyon Forms a Novel Ternary Complex with Dopamine D1 Receptor through PSD-95 Protein and Plays a Role in Dopamine Receptor Internalization

Calcyon, once known for interacting directly with the dopamine D(1) receptor (D(1)DR), is implicated in various neuropsychiatric disorders including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Although its direct interaction with D(1)DR has been shown to be misinterpreted, it still plays important roles in D(1)DR signaling. Here, we found that calcyon interacts with the PSD-95 and subsequently forms a ternary complex with D(1)DR through PSD-95. Calcyon is phosphorylated on Ser-169 by the PKC activator phorbol 12-myristate 13-acetate or by the D(1)DR agonist SKF-81297, and its phosphorylation increases its association with PSD-95 and recruitment to the cell surface. Interestingly, the internalization of D(1)DR at the cell surface was enhanced by phorbol 12-myristate 13-acetate and SKF-81297 in the presence of calcyon, but not in the presence of its S169A phospho-deficient mutant, suggesting that the phosphorylation of calcyon and the internalization of the surface D(1)DR are tightly correlated. Our results suggest that calcyon regulates D(1)DR trafficking by forming a ternary complex with D(1)DR through PSD-95 and thus possibly linking glutamatergic and dopamine receptor signalings. This also raises the possibility that a novel ternary complex could represent a potential therapeutic target for the modulation of related neuropsychiatric disorders.     

Stimulus Bias Provides Evidence for Conformational Constraints in the Structure of a G Protein-coupled Receptor

A key characteristic of G protein-coupled receptors (GPCRs) is that they activate a plethora of signaling pathways. It is now clear that a GPCR coupling to these pathways can be regulated selectively by ligands that differentially drive signaling down one pathway in preference to another. This concept, termed stimulus bias, is revolutionizing receptor biology and drug discovery by providing a means of selectively targeting receptor signaling pathways that have therapeutic impact. Herein, we utilized a novel quantitative method that determines stimulus bias of synthetic GPCR ligands in a manner that nullifies the impact of both the cellular background and the “natural bias” of the endogenous ligand. By applying this method to the M₂ muscarinic acetylcholine receptor, a prototypical GPCR, we found that mutation of key residues (Tyr-80^2.61 and Trp-99^3.28) in an allosteric binding pocket introduces stimulus bias in response to the atypical ligands AC-42 (4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)piperidine HCl) and 77-LH-28-1 (1-(3-(4-butyl-1-piperidinyl)propyl)- 3,3-dihydro-2(1H)-quinolinone). By comparing stimulus bias factors among receptor internalization, G protein activation, extracellular-regulated protein kinase 1/2 (ERK1/2) signaling, and receptor phosphorylation, we provide evidence that Tyr-80^2.61 and Trp-99^3.28 act either as molecular switches or as gatekeeper residues that introduce constraints limiting the active conformation of the M₂ muscarinic acetylcholine receptor and thereby regulate stimulus bias. Furthermore, we provide evidence that downstream signaling pathways previously considered to be related to each other (i.e. receptor phosphorylation, internalization, and activation of ERK1/2) can act independently.     

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca²⁺-ATPase (PMCA) is essential for removal of cytoplasmic Ca²⁺ and for shaping the time courses of Ca²⁺-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca²⁺ extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30''s C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other''s function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca²⁺ signaling and GPER/GPR30-mediated activities.