Extracellular loops and ligand binding to a subfamily of Family A G-protein-coupled receptors

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins which mediate their effects by coupling to G-proteins. The V1aR (V1a vasopressin receptor) is a member of a family of related GPCRs that are activated by vasopressin {AVP ([Arg8]vasopressin)}, OT (oxytocin) and related peptides. These receptors are members of a subfamily of Family A GPCRs called the neurohypophysial peptide hormone receptor family. GPCRs exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The cluster of TM helices is functionally important for ligand binding, and, furthermore, activation of GPCRs involves movement of these TM helices. Consequently, it might be assumed that the extracellular face of GPCRs is composed of peptide linkers that merely connect important TM helices. However, using a systematic mutagenesis approach and focusing on the N-terminus and the second ECL of the V1aR, we have established that these extracellular domains fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

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The third extracellular loop of G-protein-coupled receptors: more than just a linker between two important transmembrane helices

Biochemical Society Transactions ◽

10.1042/bst0321048 ◽

2004 ◽

Vol 32 (6) ◽

pp. 1048-1050 ◽

Cited By ~ 31

Author(s):

Z. Lawson ◽

M. Wheatley

Keyword(s):

G Protein ◽

Tertiary Structure ◽

Large Family ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Transmembrane Helices ◽

Ligand Selectivity ◽

Interaction Sites ◽

Amine Neurotransmitters ◽

G Protein Coupled

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins, which mediate their effects by coupling with G-proteins. Despite responding to a range of very diverse stimuli, these receptors exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The hydrophobic environment formed by the cluster of TM helices is functionally important. For example, the 11-cis retinal chromophore of rhodopsin forms a protonated Schiff base linkage to a lysine in TM7, deep within the helical bundle, and small ligands, such as amine neurotransmitters and non-peptide analogues of peptide hormones, also bind within the corresponding region of their cognate receptors. In addition, activation of GPCRs involves relative movement of TM helices to present G-protein interaction sites across the intracellular face of the receptor. Consequently, it might be assumed that the ECLs of the GPCR are inert peptide linkers that merely connect important TM helices. Focusing on ECL3 (third ECL), it is becoming increasingly apparent that this extracellular domain can fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

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A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.2041-2051.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 2041-2051 ◽

Cited By ~ 18

Author(s):

Jennifer C. Lin ◽

Ken Duell ◽

James B. Konopka

Keyword(s):

Ligand Binding ◽

G Protein ◽

Transmembrane Domain ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Transmembrane Domains ◽

Consecutive Series ◽

Subsequent Step ◽

G Protein Coupled ◽

Specific Reagent

ABSTRACT The α-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

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The Molecular Basis of G Protein–Coupled Receptor Activation

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-060614-033910 ◽

2018 ◽

Vol 87 (1) ◽

pp. 897-919 ◽

Cited By ~ 207

Author(s):

William I. Weis ◽

Brian K. Kobilka

Keyword(s):

Ligand Binding ◽

G Protein ◽

Conformational Changes ◽

Cellular Responses ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Active Conformation ◽

Allosteric Coupling ◽

External Stimuli ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

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Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation

Trends in Pharmacological Sciences ◽

10.1016/j.tips.2010.10.001 ◽

2011 ◽

Vol 32 (1) ◽

pp. 35-42 ◽

Cited By ~ 150

Author(s):

M.C. Peeters ◽

G.J.P. van Westen ◽

Q. Li ◽

A.P. IJzerman

Keyword(s):

G Protein ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Ligand Recognition ◽

Extracellular Loops ◽

G Protein Coupled

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G-protein-coupled-receptor activation of the smooth muscle calponin gene

Biochemical Journal ◽

10.1042/bj3570587 ◽

2001 ◽

Vol 357 (2) ◽

pp. 587-592 ◽

Cited By ~ 9

Author(s):

Nickolai O. DULIN ◽

Sergei N. ORLOV ◽

Chad M. KITCHEN ◽

Tatyana A. VOYNO-YASENETSKAYA ◽

Joseph M. MIANO

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

G Protein ◽

Transcriptional Activation ◽

Fold Increase ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Mitogen Activated Protein Kinase ◽

G Protein Coupled

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

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Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes

10.1101/280347 ◽

2018 ◽

Cited By ~ 2

Author(s):

Zack Zurawski ◽

Analisa D. Thompson Gray ◽

Lillian J. Brady ◽

Brian Page ◽

Emily Church ◽

...

Keyword(s):

Transgenic Mice ◽

Calcium Channels ◽

G Protein ◽

Presynaptic Inhibition ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Voltage Gated ◽

Voltage Gated Calcium Channels ◽

Crucial Component ◽

G Protein Coupled

ABSTRACTGi/o-coupled G-protein coupled receptors modulate neurotransmission presynaptically through inhibition of exocytosis. Release of Gβγ subunits decreases the activity of voltage-gated calcium channels (VGCC), decreasing excitability. A less understood Gβγ–mediated mechanism downstream of calcium entry is the binding of Gβγ to SNARE complexes. Here, we create a mouse partially deficient in this interaction. SNAP25Δ3 homozygote animals are developmentally normalbut impaired gait and supraspinal nociception. They also have elevated stress-induced hyperthermia and impaired inhibitory postsynaptic responses to α2A-AR, but normal inhibitory postsynaptic responses to Gi/o-coupled GABAB receptor activation. SNAP25Δ3 homozygotes have deficits in inhibition of hippocampal postsynaptic responses to 5 HT1b agonists that affect hippocampal learning. These data suggest that Gi/o-coupled GPCR inhibition of exocytosis through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

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A common ligand-binding site in G-protein coupled receptors

Peptides for the New Millennium - American Peptide Symposia ◽

10.1007/0-306-46881-6_229 ◽

2006 ◽

pp. 581-582

Author(s):

Laerte Oliveira ◽

Gerrit Vriend ◽

Antonio C.M. Paiva

Keyword(s):

Ligand Binding ◽

Binding Site ◽

G Protein ◽

G Protein Coupled Receptors ◽

Ligand Binding Site ◽

G Protein Coupled

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Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806077115 ◽

2018 ◽

Vol 115 (37) ◽

pp. 9252-9257 ◽

Cited By ~ 17

Author(s):

Jamie L. Lahvic ◽

Michelle Ammerman ◽

Pulin Li ◽

Megan C. Blair ◽

Emma R. Stillman ◽

...

Keyword(s):

Fatty Acids ◽

G Protein ◽

Cell Lines ◽

Saturated Fatty Acids ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Hydroxy Fatty Acids ◽

High Affinity ◽

G Protein Coupled

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

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