Receptor C-terminal Tail Research Articles

Phosphorylation patterns in the AT1R C-terminal tail specify distinct downstream signaling pathways.

Different ligands stabilize specific conformations of the angiotensin II type 1 receptor (AT1R) that direct distinct signaling cascades mediated by heterotrimeric G proteins or β-arrestin. These different active conformations are thought to engage distinct intracellular transducers because of differential phosphorylation patterns in the receptor C-terminal tail (the "barcode" hypothesis). Here, we identified the AT1R barcodes for the endogenous agonist AngII, which stimulates both G protein activation and β-arrestin recruitment, and for a synthetic biased agonist that only stimulates β-arrestin recruitment. The endogenous and β-arrestin-biased agonists induced two different ensembles of phosphorylation sites along the C-terminal tail. The phosphorylation of eight serine and threonine residues in the proximal and middle portions of the tail was required for full β-arrestin functionality, whereas phosphorylation of the serine and threonine residues in the distal portion of the tail had little influence on β-arrestin function. Similarly, molecular dynamics simulations showed that the proximal and middle clusters of phosphorylated residues were critical for stable β-arrestin-receptor interactions. These findings demonstrate that ligands that stabilize different receptor conformations induce different phosphorylation clusters in the C-terminal tail as barcodes to evoke distinct receptor-transducer engagement, receptor trafficking, and signaling.

Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods

BackgroundReplacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen.ResultsHere we generated highly soluble and stable recombinant protein constructs GST-CB1414–472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443–473 of the mouse CB1 receptor that corresponds to residues 442–472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay.ConclusionsCollectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

The P2Y2 Receptor C-Terminal Tail Modulates but Is Dispensable for β-Arrestin Recruitment.

The P2Y2 receptor (P2Y2R) is a G protein-coupled receptor that is activated by extracellular ATP and UTP, to a similar extent. This allows it to play roles in the cell’s response to the (increased) release of these nucleotides, e.g., in response to stress situations, including mechanical stress and oxygen deprivation. However, despite its involvement in important (patho)physiological processes, the intracellular signaling induced by the P2Y2R remains incompletely described. Therefore, this study implemented a NanoBiT® functional complementation assay to shed more light on the recruitment of β-arrestins (βarr1 and βarr2) upon receptor activation. More specifically, upon determination of the optimal configuration in this assay system, the effect of different (receptor) residues/regions on βarr recruitment to the receptor in response to ATP or UTP was estimated. To this end, the linker was shortened, the C-terminal tail was truncated, and phosphorylatable residues in the third intracellular loop of the receptor were mutated, in either singly or multiply adapted constructs. The results showed that none of the introduced adaptations entirely abolished the recruitment of either βarr, although EC50 values differed and time-luminescence profiles appeared to be qualitatively altered. The results hint at the C-terminal tail modulating the interaction with βarr, while not being indispensable.

Signaling at the endosome: cryo-EM structure of a GPCR-G protein-beta-arrestin megacomplex.

G protein‐coupled receptors (GPCRs) are a large class of cell‐surface receptor involved in cellular signaling that are currently the target of over one third of all clinically approved therapeutics. Classically, an agonist‐bound, active GPCR couples to and activates G proteins through the receptor intracellular core. To attenuate G protein signaling, the GPCR is phosphorylated at its C‐terminal tail and/or relevant intracellular loops, allowing for the recruitment of β‐arrestins (βarrs). βarrs then couple to the receptor intracellular core in order to mediate receptor desensitization and internalization. However, our laboratory and others have observed that some GPCRs are capable of continuously signaling through G protein even after internalization. This mode of sustained signaling stands in contrast with our previous understanding of GPCR signaling, and its molecular mechanism is still not well understood. Recently, we have solved the structure of a GPCR–G protein–βarr megacomplex by cryo‐electron microscopy. This ‘megaplex’ structure illustrates the independent and simultaneous coupling of a G protein to the receptor intracellular core, and binding of a βarr to a phosphorylated receptor C‐terminal tail, with all three components maintaining their respective canonically active conformations. The structure provides evidence for the ability of a GPCR to activate G protein even while being bound to and internalized by βarr. It also reveals that the binding of G protein and βarr to the same GPCR is not mutually exclusive, and raises a number of future questions to be answered regarding the mechanism of sustained signaling.

Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the gβ subunit.

One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular βsheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.

Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.

Acid regulation of NaDC-1 requires a functional endothelin B receptor

Metabolically generated acid is the major physiological stimulus for increasing proximal tubule citrate reabsorption, which leads to a decrease in citrate excretion. The activity of the Na-citrate cotransporter, NaDC-1, is increased in vivo by acid ingestion and in vitro by an acidic pH medium. In opossum kidney cells the acid stimulatory effect and the ability of endothelin-1 (ET-1) to stimulate NaDC-1 activity are both blocked by the endothelin B (ET(B)) receptor antagonist, BQ788. Acid feeding had no effect on brush border membrane NaDC-1 activity in mice in which ET(B) receptor expression was knocked out, whereas a stimulatory effect was found in wild-type mice. Using ET(A)/ET(B) chimeric and ET(B) C-terminal tail truncated constructs, ET-1 stimulation of NaDC-1 required a receptor C-terminal tail from either ET(A) or ET(B). The ET-1 effect was greatest when either the ET(B) transmembrane domain and C-terminal tail were present or the ET(B) C-terminal tail was linked to the ET(A) transmembrane domain. This effect was smaller when the ET(B) transmembrane domain was linked to the ET(A) C-terminal tail. Thus, the acid-activated pathway mediating stimulation of NaDC-1 activity requires a functional ET(B) receptor in vivo and in vitro, as does acid stimulation of NHE3 activity. Since increased NaDC-1 and NHE3 activities constitute part of the proximal tubule adaptation to an acid load, these studies indicate that there are similarities in the signaling pathway mediating these responses.

RXFP1 Couples to the Gαi3-Gβγ-PI3K-PKCζ Pathway via the Final 10 Amino Acids of the Receptor C-terminal Tail

The relaxin family peptide receptors RXFP1 and RXFP2 are highly similar receptors that share approximately 80% amino acid sequence homology. Constitutively active receptors couple to increased cAMP accumulation, which is important for relaxin-mediated decidualization and myometrial inhibition. Despite the high homology, the receptors couple to different G-proteins to affect cAMP accumulation. This study aimed to determine the region of RXFP1 that directs coupling to the delayed Galpha(i3) pathway by using receptor mutagenesis. Receptor chimeras suggested that activation of this pathway by RXFP1 was dependent upon the membrane-anchored domain of the receptor. Further receptor mutagenesis showed that activation of the Galpha(i3)-Gbetagamma-PI3K-PKCzeta cAMP pathway by RXFP1 is dependent upon the C-terminal 10 amino acids of the receptor and absolutely requires Arg(752).

Different abilities of the four FGFRs to mediate FGF-1 translocation are linked to differences in the receptor C-terminal tail

Members of the fibroblast growth factor family bind to one or more of the four closely related membrane-spanning FGF receptors. In addition to signaling through the receptors, exogenous FGF-1 and FGF-2 are endocytosed and translocated to the cytosol and nucleus where they stimulate RNA and DNA synthesis. Here we have studied the ability of the four FGF receptors to facilitate translocation of exogenous FGF-1 to the cytosol and nucleus. FGFR1 and FGFR4 were able to mediate translocation, whereas FGFR2 and FGFR3 completely lacked this ability. By analyzing mutant FGFRs we found that the tyrosine kinase domain could be deleted from FGFR1 without abolishing translocation, whereas the C-terminal tail of the FGFRs, constituted by approximately 50 amino acids downstream of the kinase domain, plays a crucial role in FGF-1 translocation. Three amino acids residues within the C-terminal tail were found to be of particular importance for translocation. For FGFR2, the two amino acid substitutions Q774M and P800H were sufficient to enable the receptor to support FGF-1 translocation. The results demonstrate a striking diversity in function of the four FGFRs determined by their C-terminal domain.

Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus

The recently cloned lamprey GnRH receptor was shown to have several unique features, including the longest intracellular C-terminal tail (120 amino acids (aa)) of any previously described GnRH receptor. In the current study, a series of experiments were performed examining cAMP responses, binding kinetics, whole cell competitive binding assays and internalization studies of the lamprey GnRH receptor using a series of three C-terminal tail truncations (80 aa, 40 aa and 0 aa) to better describe the functional significance of this unique vertebrate GnRH receptor. Activation of the lamprey GnRH receptor was shown to stimulate cAMP production in a dose-dependent manner when treated with either lamprey GnRH-I (LogEC50 -6.57+/-0.15) or lamprey GnRH-III (LogEC(50) -8.29+/-0.09). Truncation analysis indicated that the membrane proximal 40 aa of the lamprey GnRH receptor C-terminal tail contain a motif required for cAMP accumulation. Saturation binding assays using the wild type and truncated lamprey GnRH receptors revealed that all of three truncated lamprey GnRH receptors were capable of binding lamprey GnRH-I. Competitive, intact cell-binding assays suggested that the lamprey GnRH receptor is lamprey GnRH-III selective, based on the observed pharmacological profile: lamprey GnRH-III (Inhibitory constant (Ki) 0.708+/-0.245 nM)=chicken GnRH-II (Ki 0.765+/-0.160 nM) > mammalian GnRH (Ki 12.9+/-1.96 nM) > dAla(6)Pro(9)NEt mammalian GnRH (Ki 21.6+/-9.68 nM) > lamprey GnRH-I (Ki 118.0+/-23.6). Finally, the lamprey GnRH receptor was shown to undergo rapid ligand-dependent internalization, which was significantly diminished in the tail-less truncated form. We have shown from our current and our previous structural studies that this unique lamprey GnRH receptor shares several characteristics of both type I and type II GnRH receptors which suggests that this receptor has retained ancestral characteristics that can provide insight into the function and evolution of the vertebrate GnRH receptor family.

Effects of an α-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: Modulation by Homer

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca 2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly α-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K + channels. The peptide (0.1–10 μM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca 2+ concentration was 1 μM; but with 10 μM cytoplasmic Ca 2+, skeletal ryanodine receptors were activated by ≤1.0 μM peptide and inhibited by 10 μM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca 2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.

Periplakin Interferes with G Protein Activation by the Melanin-concentrating Hormone Receptor-1 by Binding to the Proximal Segment of the Receptor C-terminal Tail

In mice genetic ablation of expression of either melanin-concentrating hormone or the melanin-concentrating hormone-1 receptor results in alterations in energy metabolism and a lean phenotype. There is thus great interest in the function and regulation of this receptor. Using the yeast two-hybrid system we identified an interaction of the actin- and intermediate filament-binding protein periplakin with the intracellular C-terminal tail of the melanin-concentrating hormone-1 receptor. Direct association of these proteins was verified in pull-down and coimmunoprecipitation experiments. Truncations and internal deletions delineated the site of interaction to a group of 11 amino acids proximal to transmembrane helix VII, which was distinct from the binding site for the melanin-concentrating hormone-1 receptor-interacting zinc finger protein. Immunohistochemistry demonstrated coexpression of periplakin with melanin-concentrating hormone-1 receptor in specific cells of the piriform cortex, amygdala, and other structures of the adult mouse brain. Coexpression of the melanin-concentrating hormone-1 receptor with periplakin in human embryonic kidney 293 cells did not prevent agonist-mediated internalization of the receptor but did interfere with binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate ([(35)S]GTPgammaS) to the G protein Galpha(o1) and the elevation of [Ca(2+)](i). Coexpression of the receptor with the interacting zinc finger protein did not modulate receptor internalization or G protein activation. The interaction of periplakin with receptors was selective. Coexpression of periplakin with the IP prostanoid receptor did not result in coimmunoprecipitation nor interfere with agonist-mediated binding of [(35)S]GTPgammaS to the G protein Galpha(s). Periplakin is the first protein described to modify the capacity of the melanin-concentrating hormone-1 receptor to initiate signal transduction.

Oligomerization of the cardiac ryanodine receptor C-terminal tail.

The C-terminal 100 amino acids of the RyR (ryanodine receptor), referred to as the C-terminal tail, is a highly conserved sequence that is present in all known RyR isoforms and which has been implicated in channel function. Deleting the final 15 amino acids from the full-length skeletal muscle RyR resulted in an inactive channel, attributed to impaired assembly of a tetrameric RyR complex [Gao, Tripathy, Lu and Meissner (1997) FEBS Lett. 412, 223-226]. To account for these observations, the C-terminal tail itself may be an important molecular determinant of oligomerization. Alternatively, the large N-terminal cytoplasmic domain may fold back upon itself to interact with the C-terminal tail to provide a correctly folded tetrameric structure. We explored these possibilities for RyR2 (cardiac RyR) using the yeast two-hybrid interaction assay and in vitro translation followed by immunoprecipitation and chemical cross-linking. The data indicate that the C-terminal tail of RyR2 is capable of self-tetramerization. Moreover, a truncated C-terminal tail, lacking the final 15 amino acids, failed to self-associate. These observations suggest that the intrinsic ability of the RyR C-terminal tail to self-tetramerize may be vitally important for the oligomeric assembly of the native RyR channel.

Chimeric Galphaq subunits can distinguish the long form of the Xenopus Mel1c melatonin receptor from the mammalian mt1 and MT2 melatonin receptors.

The family of melatonin receptors is composed of the mt1, MT2, and Mel1c subtypes. The Mel1c is further divided into one long and two short isoforms. A recent study has shown that, unlike mt1 and MT2, the long form of Mel1c is incapable of activating the pertussis toxin-insensitive G16. Here we used three well-characterized Galphaq chimeras to explore the coupling specificity of the melatonin receptors. The qi5, qo5, and qz5 chimeras can link numerous Gi-coupled receptors to the stimulation of phosphoinositide-specific phospholipase C. Both mt1 and MT2 receptors interacted productively with the Galphaq chimeras, while the long form of Mel1c was totally ineffective. Among the Galphaq chimeras, qo5 was less efficiently coupled to the melatonin receptors. Such differential coupling is best explained by structural differences between the melatonin receptors as well as among the Galphaq chimeras. Since the long form of Mel1c receptor possesses an exceptionally large C-terminal tail, we tested the ability of four melatonin receptor C-terminal tail chimeras (Chi 1-4) to interact with the Galphaq chimeras. The presence of the large C-terminal tail of Mel1c in Chi 1 and Chi 3 markedly hindered their coupling to the Galphaq chimeras. On the other hand, the attachment of either the mtl or MT2 C-terminal tail to a Mel1c backbone produced chimeras (Chi 2 and Chi 4) that were capable of activating the Galphaq chimeras. These findings suggest the involvement of C-terminal regions of melatonin receptors in the recognition of G proteins.

Analysis of the C-terminal tail of the rat thyrotropin-releasing hormone receptor-1 in interactions and cointernalization with beta-arrestin 1-green fluorescent protein.

Coexpression of the rat thyrotropin releasing hormone receptor-1 with beta-arrestin 1-green fluorescent protein (GFP) in human embryonic kidney 293 cells results in agonist-dependent translocation of the arrestin to the plasma membrane followed by its cointernalization with the receptor. Truncations of the receptor C-terminal tail from 93 to 50 amino acids did not alter this. Truncations to fewer than 47 amino acids prevented such interactions and inhibited but did not fully eliminate agonist-induced internalization of the receptor. Deletion and site-directed mutants of the C-terminal tail indicated that separate elimination of a potential casein kinase II phosphorylation site or clathrin/clathrin adapter motifs was insufficient to prevent either internalization of the receptor or its cointernalization with beta-arrestin 1-GFP. Alteration of sites of acylation reduced internalization and prevented interactions with beta-arrestin 1-GFP. Combinations of these mutants resulted in lack of interaction with beta-arrestin 1-GFP and a 10-fold reduction in internalization of the receptor. Despite this, the receptor construct that lacked the three protein sequence motifs was fully functional. These studies map sites that contribute the interactions of the thyrotropin releasing hormone receptor-1 C-terminal tail required for effective contacts with beta-arrestin 1-GFP and indicate key roles for these interactions in agonist-induced internalization of the receptor.

Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail.

We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the beta-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr-->Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2-binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation.