GGA3 interacts with L-type prostaglandin D synthase and regulates the recycling and signaling of the DP1 receptor for prostaglandin D2 in a Rab4-dependent mechanism

Mechanisms controlling the recycling of G protein-coupled receptors (GPCRs) remain largely unclear. We report that GGA3 (Golgi-associated, γ adaptin ear containing, ADP-ribosylation factor-binding protein 3) regulates the recycling and signaling of the PGD₂ receptor DP1 through a new mechanism. An endogenous interaction between DP1 and GGA3 was detected by co-immunoprecipitation in HeLa cells. The interaction was promoted by DP1 agonist stimulation, which was supported by increased DP1-GGA3 colocalization in confocal microscopy. Pulldown assays showed that GGA3 interacts with the intracellular loop 2 and C-terminus of DP1, whereas the receptor interacts with the VHS domain of GGA3. The Arf-binding deficient GGA3 N194A mutant had the same effect as wild-type GGA3 on DP1 trafficking, suggesting a new mechanism for GGA3 in recycling. Depletion of Rab4 inhibited the GGA3 effect on DP1 recycling, revealing a Rab4-dependent mechanism. Interestingly, depletion of L-PGDS (L-type prostaglandin synthase, the enzyme that produces the agonist for DP1) impaired the ability of GGA3 to mediate DP1 recycling, while GGA3 knockdown prevented L-PGDS from promoting DP1 recycling, indicating that both proteins function interdependently. A novel interaction was observed between co-immunoprecipitated endogenous L-PGDS and GGA3 proteins in HeLa cells, and in vitro using purified recombinant proteins. Redistribution of L-PGDS towards GGA3- and Rab4-positive vesicles was induced by DP1 activation. Silencing of GGA3 inhibited ERK1/2 activation following DP1 stimulation. Altogether, our data reveal a novel function for GGA3, in a newly described association with L-PGDS, in the recycling and signaling of a GPCR, namely DP1.     

Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor

Prostaglandin D₂ (PGD₂) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gα_s-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gα_i/o proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD₂ induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.     

IQGAP1 promotes CXCR4 chemokine receptor function and trafficking via EEA-1+ endosomes

IQ motif–containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell–derived factor-1 (SDF-1; also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1⁺ endosomes, and decreased efficiency of CXCR4 recycling. SDF-1–induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to α-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1⁺ endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1⁺ endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types.     

MAGI proteins can differentially regulate the signaling pathways of 5-HT2AR by enhancing receptor trafficking and PLC recruitment

MAGI proteins are Membrane-Associated Guanylate Kinase Inverted proteins that belong to the MAGUK family. They are scaffolding proteins that were shown to mediate the trafficking and signaling of various G protein-coupled receptors (GPCRs). They contain PDZ domains in their structure and many GPCRs interact with these proteins via the PDZ motifs on the carboxyl terminal end of a receptor. In a PDZ overlay assay performed with the carboxyl terminal tail of 5-HT_2AR, we were able to detect all three members of the MAGI subfamily, MAGI-1, MAGI-2 and MAGI-3 as interacting PDZ proteins. The PDZ motif of 5-HT_2AR consists of three amino acids; serine (S), cysteine (C) and valine (V). In this study, we characterize these 5-HT_2AR interactions with MAGI proteins. We first confirm the interaction using co-immunopricipitation and illustrate that the interaction is PDZ motif-dependent in human embryonic kidney (HEK 293) cells. We then assess the effects of overexpression and knockdown of the MAGI proteins on the internalization, trafficking and signaling of 5-HT_2AR. We find that knockdown of either MAGI-1 or MAGI-3 using siRNA results in a significant reduction in the internalization of 5-HT_2AR. As for signaling, we report here that MAGI proteins can modulate the signaling via the two transduction pathways that 5-HT_2AR can activate. We illustrate a significant effect of modulating MAGI proteins expression on 5-HT-stimulated IP formation. We demonstrate an enhancement in 5-HT_2AR-stimulated IP formation upon MAGI proteins overexpression. In addition, we show that knockdown of MAGI proteins with siRNA leads to a significant reduction in 5-HT_2AR-stimulated IP formation. Furthermore, we illustrate a significant increase in 5-HT-stimulated ERK1/2 phosphorylation upon MAGI proteins knockdown. Interestingly, this effect on ERK1/2 activation is PDZ motif-independent. We also suggest two possible mechanisms of regulation for the effect of MAGI proteins on 5-HT_2AR function. One mechanism involves the regulation of cell surface expression since we show that both MAGI-2 and MAGI-3 can enhance receptor trafficking to the plasma membrane when overexpressed in HEK 293 cells. The other mechanism points to regulation of second messengers in the signaling pathways. Specifically, we show that overexpression of any of the three MAGI proteins can enhance the recruitment of PLCβ3 to 5-HT_2AR. In addition, we report a negative effect for knocking down MAGI-3 on β-arrestin recruitment to the receptor and this effect is PDZ motif-independent. Taken together, our findings document distinct roles for the three MAGI proteins in regulating 5-HT_2AR trafficking and signaling and emphasize the importance of studying PDZ proteins and their interactions with GPCRs to regulate their function.     

A G protein–coupled receptor and the intracellular synthase of its agonist functionally cooperate

Export of newly synthesized G protein–coupled receptors (GPCRs) remains poorly characterized. We show in this paper that lipocalin-type prostaglandin D₂ (PGD₂) synthase (L-PGDS) interacts intracellularly with the GPCR DP1 in an agonist-independent manner. L-PGDS promotes cell surface expression of DP1, but not of other GPCRs, in HEK293 and HeLa cells, independent of L-PGDS enzyme activity. In addition, formation of a DP1–Hsp90 complex necessary for DP1 export to the cell surface is dependent on the interaction between L-PGDS and the C-terminal MEEVD residues of Hsp90. Surprisingly, PGD₂ synthesis by L-PGDS is promoted by coexpression of DP1, suggesting a possible intracrine/autocrine signaling mechanism. In this regard, L-PGDS increases the formation of a DP1–ERK1/2 complex and increases DP1-mediated ERK1/2 signaling. Our findings define a novel cooperative mechanism in which a GPCR (DP1) promotes the activity of the enzyme (L-PGDS) that produces its agonist (PGD₂) and in which this enzyme in turn acts as a cofactor (of Hsp90) to promote export and agonist-dependent activity of the receptor.     

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α₂-adrenergic receptors (α₂-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α_2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α_2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α_2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α_2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.     

Metabolic depletion of sphingolipids inhibits agonist-induced endocytosis of the serotonin1A receptor

G protein-coupled receptors (GPCRs) are vital cellular signaling machinery and currently represent ~40% drug targets. Endocytosis of GPCRs is an important process that allows stringent spatiotemporal control over receptor population on the cell surface. Although the role of proteins in GPCR endocytosis is well addressed, the contribution of membrane lipids in this process is rather unexplored. Sphingolipids are essential functional lipids in higher eukaryotes and are implicated in several neurological functions. To understand the role of sphingolipids in GPCR endocytosis, we subjected cells expressing human serotonin_1A receptors (an important neurotransmitter GPCR involved in cognitive and behavioral functions) to metabolic sphingolipid depletion using fumonisin B₁, an inhibitor of sphingolipid biosynthetic pathway. Our results, using flow cytometric analysis and confocal microscopic imaging, show that sphingolipid depletion inhibits agonist-induced endocytosis of the serotonin_1A receptor in a concentration-dependent manner, which was restored when sphingolipid levels were replenished. We further show that there was no change in the internalization of transferrin, a marker for clathrin-mediated endocytosis, under sphingolipid-depleted condition, highlighting the specific requirement of sphingolipids for endocytosis of serotonin_1A receptors. Our results reveal the regulatory role of sphingolipids in GPCR endocytosis and highlight the importance of neurotransmitter receptor trafficking in health and disease.     

Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signaling

The serotonin (5-hydroxytryptamine; 5-HT)_2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT_2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca²⁺-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT_2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT_2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT_2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT_2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT_2C receptor agonists and their antidepressant-like activity.     

Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport

The molecular mechanisms underlying the anterograde surface transport of G protein–coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α_2A-adrenergic receptor (α_2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α_2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER–Golgi-surface transport of α_2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β₂-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α_2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.     

IQGAP1 Regulates Endothelial Barrier Function via EB1-Cortactin Cross Talk

Cross talk between the actin cytoskeleton and microtubules (MT) has been implicated in the amplification of agonist-induced Rho signaling, leading to increased vascular endothelial permeability. This study tested the involvement of actin-MT cross talk in the mechanisms of barrier enhancement induced by hepatocyte growth factor (HGF) and evaluated the role of the adaptor protein IQGAP1 in integrating the MT- and actin-dependent pathways of barrier enhancement. IQGAP1 knockdown by small interfering RNA attenuated the HGF-induced increase in endothelial barrier properties and abolished HGF-activated cortical actin dynamics. IQGAP1 reduction abolished HGF-induced peripheral accumulation of Rac cytoskeletal effector cortactin and cortical actin remodeling. In addition, HGF stimulated peripheral MT growth in an IQGAP1-dependent fashion. HGF also induced Rac1-dependent IQGAP1 association with the MT fraction and the formation of a protein complex containing end-binding protein 1 (EB1), IQGAP1, and cortactin. Decreasing endogenous IQGAP1 abolished HGF-induced EB1-cortactin colocalization at the cell periphery. In turn, expression of IQGAP1ΔC (IQGAP1 lacking the C-terminal domain) attenuated the cortactin association with EB1 and suppressed HGF-induced endothelial cell peripheral actin cytoskeleton enhancement. These results demonstrate for the first time the MT-actin cross talk mechanism of HGF-induced endothelial barrier enhancement and suggest that IQGAP1 functions as a hub linking HGF-induced signaling to MT and actin remodeling via EB1-IQGAP1-cortactin interactions.     

Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor

Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y₆. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y₆ receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y₆. Interestingly, UDP induced clathrin-dependent P2Y₆ internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y₆ was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y₆ internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y₆ receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y₆ signaling.     

Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling

The special glycerophospholipids plasmalogens (Pls) are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor) proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh)-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.     

The changing world of G protein-coupled receptors: from monomers to dimers and receptor mosaics with allosteric receptor–receptor interactions

Based on indications of direct physical interactions between neuropeptide and monoamine receptors in the early 1980s, the term receptor–receptor interactions was introduced and later on the term receptor heteromerization in the early 1990s. Allosteric mechanisms allow an integrative activity to emerge either intramolecularly in G protein-coupled receptor (GPCR) monomers or intermolecularly via receptor–receptor interactions in GPCR homodimers, heterodimers, and receptor mosaics. Stable heteromers of Class A receptors may be formed that involve strong high energy arginine–phosphate electrostatic interactions. These receptor–receptor interactions markedly increase the repertoire of GPCR recognition, signaling and trafficking in which the minimal signaling unit in the GPCR homomers appears to be one receptor and one G protein. GPCR homomers and GPCR assemblies are not isolated but also directly interact with other proteins to form horizontal molecular networks at the plasma membrane.     

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to G_i, G_q and G₁₂ to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gα_q/11, and PAR1-RGS4 by Gα_o, but not by other Gα subunits. Gα_q/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-G_q/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.     

Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the δ-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala², D-Leu⁵]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH₃ revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-δ, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.     

Design and biological assessment of membrane-tethering neuroprotective peptides derived from the pituitary adenylate cyclase-activating polypeptide type 1 receptor

BackgroundThe pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs. Thus, we hypothesized that peptides derived from the ICLs (called pepducins) of PAC1 might initiate, as allosteric ligands, signaling cascades after recognition of the parent receptor and modulation of its conformational landscape.MethodsThree pepducins were synthesized and evaluated for their ability to 1) promote cell survival; 2) stimulate various signaling pathways associated with PAC1 activation; 3) modulate selectively PAC1, VPAC1 or VPAC2 activation; and 4) sustain mobility and prevent death of dopaminergic neurons in a zebrafish model of neurodegeneration.ResultsAssays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gα_s and Gα_q, with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gα_s stimulation. Finally, experiments, using a zebrafish neurodegeneration model, showed a neuroprotective action with all three pepducins and in particular, revealed the ability of PAC1-Pep1 and PAC1-Pep3 to preserve fish mobility and tyrosine hydroxylase expression in the brain.ConclusionWe have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR.General significancePAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules.