Protein-coupled Receptor Associated Sorting Protein Research Articles

GPRASP2 deficiency contributes to apoptosis in the spiral ganglion cells via the AMPK/DRP1 signaling pathway

G protein-coupled receptor-associated sorting protein 2 (GPRASP2) deficiency has been implicated in immunological inflammation, cancers, and neurological disorders. Our previous work revealed that the pathogenic mutation in GPRASP2 was responsible for X-linked recessive syndromic hearing loss (SHL). Given the specific high expression of GPRASP2 in the spiral ganglion, GPRASP2 likely contributes to the maintenance and functionality of neurons, potentially playing a role in synaptic transmission. The impact of GPRASP2 deficiency on spiral ganglion cells (SGCs) and their underlying pathogenic mechanisms will be investigated in this study. The primary culture of SGCs obtained from mouse cochleae was treated with Gprasp2-targeting short hairpin RNA (Gprasp2-shRNA) via lentivirus infection. The results showed that GPRASP2 deficiency enhanced SGCs apoptosis and decreased cell viability. Meanwhile, a significant abnormality of mitochondrial morphology and decreased membrane potential were observed in GPRASP2-deficient SGCs. These effects could be mitigated by treatment with the mitochondrial division inhibitor 1 (Mdivi-1). In addition to enhancing SGCs apoptosis and decreasing cell viability, GPRASP2 deficiency also inhibited the development of SGCs in mouse cochlear explant culture. Our study further revealed that this deficiency resulted in increased phosphorylation of AMPK and activation of the AMPK/DRP1 pathway, promoting SGCs apoptosis. These findings provide insight into the pathogenic mechanisms by which GPRASP2 deficiency is implicated in auditory dysfunction.

Characterization of anti-GASP motif antibodies that inhibit the interaction between GPRASP1 and G protein-coupled receptors

G protein-coupled receptor associated sorting protein 1 (GPRASP1) belongs to a family of 10 proteins that display sequence homologies in their C-terminal region. Several members including GPRASP1 also display a short repeated sequence called the GASP motif that is critically involved in protein-protein interactions with G protein-coupled receptors (GPCRs). Here, we characterized anti-GASP motif antibodies and investigated their potential inhibitory functions. We first showed that our in-house anti-GPRASP1 rabbit polyclonal serum contains anti-GASP motif antibodies and purified them by affinity chromatography. We further showed that these antibodies can detect GPRASP1 and GPRASP2 in Western blot, immunoprecipitation and immunofluorescence experiments while a mutant of GPRASP2, in which the most conserved hydrophobic core of the GASP motifs is mutated, was no more detected. Further characterization of anti-GASP motif antibodies by ELISA and Surface Plasmon Resonance assays suggests that GASP motifs function as multivalent epitopes. Finally, we set-up an Amplified Luminescent Proximity Homogeneous AlphaScreen® assay to detect the interaction between purified ADRB2 receptor and the central domain of GPRASP1 and showed that anti-GASP motif antibodies efficiently inhibit this interaction. Altogether, our results suggest that anti-GASP motif antibodies could represent a valuable tool to neutralize the interaction of GPRASP1 and GPRASP2 with different GPCRs.

Disruption of Gprasp2 down-regulates Hedgehog signaling and leads to apoptosis in auditory cells

GPRASP2 is implicated in nervous system diseases, tumors and immune inflammation. In our previous study, G protein-coupled receptor associated sorting protein 2 (GPRASP2) was identified as a novel causal gene for X-linked recessive syndromic hearing loss (SHL). However, the role of GPRASP2 in auditory function has not been elucidated. The Gprasp2-knockout (KO) mouse HEI-OC1 auditory cells were constructed using CRISPR/Cas9-mediated gene editing. RNA-sequencing (RNA-seq) was used to investigate the differentially expressed genes (DEGs) and DEGs-enriched signaling pathways, which was verified by Western blot. Flow cytometry assay was used to examine cell apoptosis. The cytological pathology was evaluated by laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). Mitochondrial damage was observed in Gprasp2-KO HEI-OC1 cells. RNA-seq analysis suggested that Gprasp2-KO was implicated in the apoptosis process, which could be mediated by Hedgehog (Hh) signaling pathway. The key molecules in Hh signaling pathway (Smo, Gli1, Gli2) were detected to be down-regulated in Gprasp2-KO HEI-OC1 cells. The differential expression of apoptosis molecules (Bcl2, Bax, Caspase-3/cleaved-Caspase-3) indicated that Gprasp2-KO induced apoptosis in HEI-OC1 cells. The treatment of smoothened agonist (Purmorphamine, PUR) activated the Hh-Gli signaling pathway and reduced apoptosis in Gprasp2-KO HEI-OC1 cells. This study revealed that Gprasp2-disruption inhibited Hh signaling pathway and led to cell apoptosis in HEI-OC1 cells, which might provide the potential molecular mechanism of GPRASP2 mutation associated with human SHL.

G protein-coupled receptor-associated sorting proteins: function and relevant disorders.

G protein-coupled receptors (GPCRs), the largest family of membrane protein receptors, can be activated by a variety of ligands and participate in signaling transduction, and they are essential in the physiologic process in vivo. GPCR-associated sorting proteins (GASPs) play an important role in the post-endocytic sorting of GPCRs. They mediate the degradation or recycling pathway, and regulate cell signaling transduction and other biological processes. The functional defects of GASPs have been reported to be implicated in pathogenesis of some neurological diseases, tumors and deafness and so on. In this review, we summarize the GASPs' function, GPCR-GASP interactions, GPCR sorting pathway and GASP-related signaling pathways implicated in the transcriptional regulation. It could help to understand the potential linkage between GASPs' dysfunction and diseases, and provide a new approach and strategy for the treatment of GASP-related diseases.

155 Evaluation of cytokine response as an indicator of genetic resistance to fescue toxicosis in purebred Angus cattle

Abstract Fescue toxicosis is a multifaceted syndrome common in cattle grazing endophyte-infected tall fescue. However, varying responses among cattle potentially implies genetic tolerance to the syndrome. Therefore, the objective was to evaluate the cytokine response of cows deemed either tolerant or susceptible to endophyte-infected fescue. Angus cows, 2–4 yrs old, grazed endophyte-infected tall fescue at two locations for 13 weeks starting mid-April 2016. A post-hoc analysis of animal performance was utilized to designate cattle into either tolerant (n = 20) or susceptible (n = 20) groups, based on physiological responses (changes in ADG) to chronic exposure to endophyte-infected fescue. Blood samples collected on weeks 1, 7, and 13 were used to evaluate serum prolactin (PRL) concentration. Cytokine concentrations were evaluated via Quantibody Bovine Cytokine Arrays. Data were analyzed using PROC MIXED of SAS with repeated measures. There were no differences by location or week. Tolerant cows tended to have decreased overall PRL concentrations compared to susceptible cows (98.3 and 138.4 ng/mL, respectively; P = 0.08). G protein-coupled receptor associated sorting protein (GASP)-1, a down-regulator of G protein-coupled receptors including D2-dopamine receptor a regulator of PRL secretion, was greater in tolerant cows when compared to susceptible cows (4.7 and 4.2 ng/mL; P = 0.03). Vascular endothelial growth factor (VEGF)-A was greater in tolerant cows when compared to susceptible cows (0.5 and 0.2 ng/mL; P = 0.02). Finally, interleukin (IL)-4, an anti-inflammatory cytokine, tended to be greater in tolerant cows when compared to susceptible cows (2.2 and 0.8 ng/mL, respectively; P = 0.07). Based on these data, greater concentrations of GASP-1, VEGF-A, and IL-4 in tolerant compared to susceptible cattle may mitigate the detrimental effects of fescue toxicosis, including suppressed PRL and vasoconstriction, leading to improved animal performance in a fescue environment. Additionally, these cytokines may serve as potential biomarkers for determining an animal’s tolerance or susceptibility to negative effects associated with endophyte-infected tall fescue

Dynamic expression analysis of armc10, the homologous gene of human GPRASP2, in zebrafish embryos.

G protein-coupled receptor-associated sorting protein 2 (GPRASP2), a member of the GASP family, has been reported to be involved in the modulation of transcription. However, few studies have revealed the role of GPRASP2 in the development and progression of diseases. As a model organism, zebrafish have been widely used to investigate human diseases. In the present study, zebrafish armadillo repeat-containing 10 (armc10), an orthologous gene of human GPRASP2 was identified, and the spatial and temporal expression patterns of armc10 in zebrafish during early embryonic development were revealed. Bioinformatics analyses showed that ARMC10 protein sequences were highly conserved. Reverse transcription polymerase chain reaction analysis and whole mount in situ hybridization revealed that zebrafish armc10 was maternally expressed and was detected at a weak level up to 12 h post-fertilization (hpf), however, its expression increased to a high level at 24 hpf. At the 75% epiboly stage and 12 hpf, armc10 was widely expressed in the embryo. At 24 hpf, armc10 mRNA was expressed in the nervous system of the zebrafish head. When the embryo was 2 days old, the wide expression of armc10 was maintained in the nervous system of the zebrafish head. At 72 hpf, the mRNA expression of armc10 was located specifically in the otic vesicles in addition to the nervous system of the head. At 96 hpf, the expression of armc10 was maintained in the otic vesicles and the nervous system of the head. The results of the present study provided novel insight into the spatial and temporal mRNA expression of armc10 in zebrafish, for the further investigation of nervous system diseases.

Novel small molecules targeting ciliary transport of Smoothened and oncogenic Hedgehog pathway activation.

Trafficking of the G protein-coupled receptor (GPCR) Smoothened (Smo) to the primary cilium (PC) is a potential target to inhibit oncogenic Hh pathway activation in a large number of tumors. One drawback is the appearance of Smo mutations that resist drug treatment, which is a common reason for cancer treatment failure. Here, we undertook a high content screen with compounds in preclinical or clinical development and identified ten small molecules that prevent constitutive active mutant SmoM2 transport into PC for subsequent Hh pathway activation. Eight of the ten small molecules act through direct interference with the G protein-coupled receptor associated sorting protein 2 (Gprasp2)-SmoM2 ciliary targeting complex, whereas one antagonist of ionotropic receptors prevents intracellular trafficking of Smo to the PC. Together, these findings identify several compounds with the potential to treat drug-resistant SmoM2-driven cancer forms, but also reveal off-target effects of established drugs in the clinics.

Abstract P264: Association of D-dimer with Incident Cardiovascular Disease Events and F3 Gene Variants in African Americans: Results From the Jackson Heart Study

Plasma D-dimer, a fibrin degradation product, is a biomarker of both coagulation and fibrinolysis and a risk factor for both arterial and venous thrombosis. African Americans (AAs) have increased risk of thrombosis and higher levels of D-dimer as compared to European populations, but little is known about the genetic architecture of D-dimer in AAs. D-dimer was assessed in 4163 AAs from the Jackson Heart Study (mean age 55 years, 62.1% female). We hypothesized that D-dimer levels would associate with incident cardiovascular disease events and with both coding and noncoding genetic variants. Median (interquartile range) D-dimer in the cohort was 0.40 (0.26-0.64) μg/ml. D-dimer was associated with increased risk of coronary heart disease (hazard ratio (HR) per standard deviation (SD)=1.26, p=0.012), stroke (HR=1.27, p=0.018), heart failure (HR=1.33, p=0.001), and mortality (HR=1.25, p<0.0001) in models adjusted for age, sex, BMI, current smoking, diabetes, hypertension, systolic blood pressure, and low-density lipoprotein cholesterol. Using data from the Affymetrix Genome-Wide Human SNP Array 6.0, imputation based on the 1000 Genomes project reference panel (Phase I, Version 3, March 2012 release) was completed using MACH 1.0 and minimac in 2709 participants with D-dimer. Associations were assessed using MACH2QTL v.1.13, with models adjusted for age, sex, and the first ten principal components for global ancestry calculated using EIGENSOFT. The most significant signal was for an intergenic locus near F3 (top variant rs2022030, p=5.71x10 -12 , minor allele frequency=10.6%, imputation r 2 =1). F3 codes for tissue factor, a necessary co-factor for factor VIIa in the initiation of the extrinsic blood coagulation pathway. The F3 locus had been previously associated with D-dimer levels in European populations but has not been reported for AAs. Data were also available from the Illumina HumanExome Beadchip, version 1.1, in 2502 individuals; single variant analyses and gene-based tests for nonsynonymous, splicing, and nonsense variants with a minor allele frequency <5% were performed using RAREMETAL, adjusted for age, sex, and the first ten principal components. For single variant analyses, a nonsynonymous variant in GPRASP1 (G protein-coupled receptor associated sorting protein 1, rs145805077, p= 1.84x10 -7 , minor allele frequency=0.3%) was significantly associated with decreased D-dimer. No gene-based test result met the strict multiple comparison significance threshold; the top gene-based result with the sequence kernel association test (SKAT) was for six variants in PPM1M (protein phosphatase, Mg2+/Mn2+ dependent, 1M, p=6.09x10 -6 ). These results highlight D-dimer as an important predictor of cardiovascular disease risk in AAs and that levels of this biomarker are associated with the F3 locus.

Pitchfork and Gprasp2 Target Smoothened to the Primary Cilium for Hedgehog Pathway Activation.

The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation.

The G Protein-Coupled Receptor Associated Sorting Proteins, Gprasp2 and Armcx1 Are Putative Negative Regulators of HSC Engraftment and Repopulation

Although hematopoietic stem cell transplantation (HSCT) is used routinely to cure hematologic disease, the efficacy of transplantation is limited by the paucity of HSC. One way to overcome this is to increase the efficiency of HSC engraftment. Thus, we executed a functional screen for novel regulators of HSCT. Murine HSC were lentivirally transduced with shRNAs targeting prioritized gene candidates prior to transplantation into cohorts of lethally irradiated recipient mice. In total, around 1300 mice were transplanted to assess a putative role for about 50 genes in HSCT. We thereby identified Gprasp2 and Armcx1 as putative negative regulators of HSCT. When transplanted at a 1:4 disadvantage relative to control, recipients of either Gprasp2 or Armcx1 shRNA-treated CD45.2 Lineage- Sca-1+ c-Kit+ (LSK) cells displayed 3.12 (p=0.024) and 2.8 (p=0.04) fold enhanced CD45.2 chimerism in peripheral blood (PB) at 16 weeks post-transplant, respectively, relative to mice transplanted with CD45.2 LSK cells treated with control shRNAs. Although loss of each gene did not favor a particular PB lineage, CD45.2 chimerism was enhanced in all bone marrow (BM) HSC and progenitor (HSPC) compartments in these recipients, correlating with their enhanced PB chimerism. qRT-PCR reveals that both murine Armcx1 and Gprasp2 are highly enriched for expression in LSK CD150+CD48- cells relative to all downstream hematopoietic progeny. Further, HemaExplorer, a bioinformatics database of human hematopoietic gene expression, suggests that GPRASP2 and ARMCX1 are also highly expressed in human HSC. This prediction is currently being validated by qRT-PCR. Interestingly, Gprasp2 and Armcx1 both belong to the G protein-coupled receptor associated sorting protein (GASP) gene family, which has never before been implicated in HSC function. The closely related GASP family member, Gprasp1, sorts G protein-coupled receptors (GPCR) to lysosomes for degradation. As Gprasp1 and Gprasp2 both contain GPCR-binding domains and ~70% amino acid sequence conservation in their C-termini, Gprasp2 may also regulate GPCR trafficking and degradation in HSC. Although Gprasp1 was not tested in our screen, qRT-PCR analysis reveals that it is also highly expressed by murine HSC relative to downstream progeny, suggesting that it too may play a role in HSC function. We are currently assessing this using Gprasp1-shRNAs and competitive transplantation. In contrast, Armcx1 lacks the GPCR binding domain and contains both a nuclear and mitochondrial localization signal and has been shown to localize to mitochondrial networks when expressed in HEK-293 cells, suggesting a role in mitochondrial/nuclear communication. To determine how loss of Gprasp2 and Armcx1 promotes HSC engraftment, we are currently employing transplantation and ex vivo culture assays to analyze the effect of their loss on cell cycle, apoptosis, migration, and adhesion of HSPC post-transplant. Our work may help elucidate the mechanisms underlying efficient engraftment, adhesion, and retention of HSPC in the BM niche, which in turn may shed light on novel pathways that could be targeted to promote the efficiency of HSCT in the clinic. DisclosuresNo relevant conflicts of interest to declare.

The G protein-coupled receptor-associated protein 1 (GASP-1) regulates rimonabant-induced downregulation of GPR55

Background The G protein-coupled receptor 55 (GPR55) has recently been suggested to be responsible for those cannabinoid responses that could not be attributed to either the cannabinoid 1 (CB1) or cannabinoid 2 (CB2) receptor. Several potent GPR55 agonists were identified such as lysophosphatidylinositol (LPI) and several synthetic cannabinoids: One of these is rimonabant (SR141716A), an antagonist at the CB1 receptor, which showed clinical promise, but approval was revoked due to adverse events. Generally, the activity of G protein coupled receptors (GPCRs) is coordinated by receptor signalling, receptor desensitization and receptor resensitization. One regulatory mechanism to guarantee appropriate GPCR expression levels in physiological conditions is that of downregulating GPCRs via the G protein-coupled receptor-associated sorting protein 1 (GASP-1), thus leading to an attenuation of cellular signalling events. GASP-1 was originally found to target δ opioid receptors to lysosomes and, hence, to the degradative pathway. It was shown that GASP-1 is a key determinant in the development of analgesic tolerance to cannabinoids via its role in facilitating downregulation of the CB1 receptor.

The trafficking of GPR55 is regulated by the G protein-coupled receptor-associated sorting protein 1

Background The G protein-coupled receptor 55 (GPR55) has recently been suggested to be responsible for those cannabinoid responses that could not be attributed to either the cannabinoid 1 (CB1) or cannabinoid 2 (CB2) receptor. Several potent GPR55 agonists were identified, such as lysophosphatidylinositol (LPI) and synthetic cannabinoids: One of these is rimonabant (SR141716A), which until that date was known to be an inverse agonist/ antagonist on the CB1 receptor. Rimonabant has further attracted attention since it was marketed to induce weight loss and reduce smoking. However, due to severe side effects after prolonged use, such as the development of anxiety and depression, rimonabant was taken off the market. Generally, the activity of GPCRs is coordinated by receptor signaling, receptor desensitization and receptor resensitization. One regulatory mechanism to guarantee appropriate GPCR expression levels in physiological conditions is that of downregulating GPCRs via the G protein-coupled receptor-associated sorting protein 1 (GASP-1), thus leading to an attenuation of cellular signaling events. GASP-1 was originally found to target δ opioid receptors to lysosomes and, hence, to be a degradative pathway. It was shown that GASP-1 is a key determinant in the development of analgesic tolerance to cannabinoids via its role in facilitating downregulation of the CB1 receptor.

The G protein-coupled receptor associated sorting protein GASP-1 regulates the signaling and trafficking of the viral chemokine receptor US28

Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Interactions of the G protein-coupled receptor-associated sorting proteins (GASP) 1 and 2 with the novel cannabinoid receptor GPR55

GPR55 was recently found to be a novel cannabinoid receptor and a member of the G protein-coupled receptor (GPCR) subfamily A. GPR55 is activated by different cannabinoid ligands as well as by the lipid ligand lysophosphatidylinositol (LPI). Generally, the activity of GPCRs is coordinated by receptor signaling, receptor desensitization and receptor resensitization. The latter two mechanisms are typically associated with the sorting of the GPCRs between degradation or recycling pathways and are highly regulated. Several sorting proteins have recently been identified, for example the G protein-coupled receptor-associated sorting protein-1 (GASP-1). GASP-1 was originally found to target delta opioid receptors (DORs) to lysosomes and hence a degradative pathway. This study shows that GPR55 can internalize after ligand activation and is subsequently targeted to intracellular vesicles of the lysosomal compartments. This result and the close similarity of GPR55 to the cannabinoid receptor CB1 – which is targeted to lysosomes via the GASP-1 protein – suggested that GASP may be involved in targeting GPR55 to lysosomes. In fact, here we show that the C-terminus of GPR55 binds GASP-1, cGASP-1 (the C-terminal part of GASP-1) and GASP-2 (the closest homologue to GASP-1) in vitro. Both GASP-1 and cGASP-1 show a high affinity to the C-terminus of GPR55 in vitro, with a ~2-fold higher affinity for GASP-1 when compared to the DOR. Interestingly, the GASP-2 isoform has the highest affinity for GPR55 with ~85% when compared to GASP-1 and cGASP-1 (~80%). This study provides first evidence that the novel cannabinoid receptor GPR55 is targeted to lysosomes after prolonged agonist stimulation and this mechanism is likely regulated by members of the newly discovered G protein-coupled receptor-associated sorting proteins, i.e GASP-1 and GASP-2.

US28 in melanoma growth

The human cytomegalovirus (hCMV) encodes the G protein-coupled receptor (vGPCR) US28. This receptor signals constitutively and interacts with a broad range of chemokines, which are crucial to the pathophysiological significance and immunoregulatory aspects of this receptor. Chemokines and their receptors have been shown to be key determinants of tumor growth and formation of metastases. US28 exerts anti-tumorigenic effects in two melanoma cell lines. Here we show that in contrast to all other previously studied cell lines, US28 is expressed on the cell surface in the melanoma cell lines Sbcl2 and 451Lu. We found that GASP – the G protein-coupled receptor-associated sorting protein – which sorts US28 and many other GPCRs to the lysosomes is absent in melanoma cells. The absence of GASP might affect the tumor-suppressing properties of US28. Melanoma cells produce several chemokines, one of them is the monocyte chemoattractant protein-1 (MCP-1). We found that the MCP-1 level in the supernatants of 451Lu cells increased when US28 wt was expressed. US28Δ317, a truncated mutant of US28 which is known to have enhanced signalling capacity compared to US28 wt, increased the MCP-1 concentration up to 20-fold. We speculate that the constitutive signalling of the receptor regulates the MCP-1 production of melanoma cells.

Interactions of the G protein-coupled receptor-associated sorting proteins (GASP) 1 and 2 with the novel cannabinoid receptor GPR55

Interactions of the G protein-coupled receptor-associated sorting proteins (GASP) 1 and 2 with the novel cannabinoid receptor GPR55

The hCMV chemokine receptor US28 prevents melanoma growth

The human cytomegalovirus (hCMV) encodes the G protein-coupled receptor (vGPCR) US28. This receptor signals constitutively and interacts with a broad range of chemokines, which are crucial to the pathophysiological significance and immunregulatory aspects of this receptor. Chemokines and their receptors have been shown to be key determinants of tumor growth and formation of metastases. US28 (and mutants thereof, i.e. US28R129A and US28Δ317) exert anti-tumorigenic effects in various melanoma cell lines by scavenging chemokines from the tumor environment. Here we show that in contrast to all other previously studied cell lines, US28 is expressed on the cell surface in the melanoma cell lines Sbcl2 and 451Lu. We suggest that GASP – the G protein-coupled receptor-associated sorting protein – which sorts US28 and many other GPCRs to the lysosomes is absent in melanoma cells. The absence of GASP might effect the tumor suppressing properties of US28. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Huntingtin interacts with the receptor sorting family protein GASP2

Protein interaction networks are useful resources for the functional annotation of proteins. Recently, we have generated a highly connected protein-protein interaction network for Huntington's disease (HD) by automated yeast two-hybrid (Y2H) screening (Goehler et al., 2004). The network included several novel direct interaction partners for the disease protein huntingtin (htt). Some of these interactions, however, have not been validated by independent methods. Here we describe the verification of the interaction between htt and GASP2 (G protein-coupled receptor associated sorting protein 2), a protein involved in membrane receptor degradation. Using membrane-based and classical coimmunoprecipitation assays we demonstrate that htt and GASP2 form a complex in cotransfected mammalian cells. Moreover, we show that the two proteins colocalize in SH-SY5Y cells, raising the possibility that htt and GASP2 interact in neurons. As the GASP protein family plays a role in G protein-coupled receptor sorting, our data suggest that htt might influence receptor trafficking via the interaction with GASP2.

A Library of 7TM Receptor C-terminal Tails: INTERACTIONS WITH THE PROPOSED POST-ENDOCYTIC SORTING PROTEINS ERM-BINDING PHOSPHOPROTEIN 50 (EBP50), N-ETHYLMALEIMIDE-SENSITIVE FACTOR (NSF), SORTING NEXIN 1 (SNX1), AND G PROTEIN-COUPLED RECEPTOR-ASSOCIATED SORTING PROTEIN (GASP)

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in post-endocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK(1) receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor.

Identification of a novel family of G protein-coupled receptor associated sorting proteins.

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.