Galectin-8 modulates human osteoclast activity partly through isoform-specific interactions.

In overactive human osteoclasts, we previously identified an alternative splicing event in LGALS8, encoding galectin-8, resulting in decreased expression of the long isoform. Galectin-8, which modulates cell-matrix interactions and functions intracellularly as a danger recognition receptor, has never been associated with osteoclast biology. In human osteoclasts, inhibition of galectin-8 expression revealed its roles in bone resorption, osteoclast nuclearity, and mTORC1 signaling regulation. Galectin-8 isoform-specific inhibition asserted a predominant role for the short isoform in bone resorption. Moreover, a liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis of galectin-8 isoforms performed in HEK293T cells identified 22 proteins shared by both isoforms. Meanwhile, nine interacting partners were specific for the short isoform, and none were unique to the long isoform. Interactors specific for the galectin-8 short isoform included cell adhesion proteins and lysosomal proteins. We confirmed the interactions of galectin-8 with CLCN3, CLCN7, LAMP1, and LAMP2, all known to localize to secretory vesicles, in human osteoclasts. Altogether, our study reveals direct roles of galectin-8 in osteoclast activity, mostly attributable to the short isoform.

Distribution of delta and mu opioid receptor mRNA in rodent dorsal root ganglia neurons.

Primary afferents are responsible for transmitting signals produced by noxious stimuli from the periphery to the spinal cord. Mu and delta opioid receptors (MOP and DOP) have analgesic properties and are highly expressed in dorsal root ganglia (DRG) neurons. In humans, spinal DOP is almost exclusively located on central terminals of DRG neurons, whereas in rodents, it is expressed both on presynaptic terminals and spinal neurons. In this study, we aimed to assess the distribution of MOP and DOP in the DRGs of mice and rats. Using in situ hybridization and immunofluorescence, we visualized MOP and DOP mRNA together with various neuronal markers. In rats and mice, we show that both receptors are expressed, albeit to different extents, in all types of neurons, namely, large and medium myelinated neurons (NF200-positive), small nonpeptidergic (IB4- or P2X3R-positive) and peptidergic C fibres (Tac1-positive). Overall, DOP mRNA was found to be mainly expressed in large and medium myelinated neurons, whereas MOP mRNA was mainly found in C fibres. The distribution of MOP and DOP, however, slightly differs between rats and mice, with a higher proportion of small nonpeptidergic C fibres expressing DOP mRNA in mice than in rats. We further found that neither morphine nor inflammation affected the distribution of the receptor mRNA. Because of their location, our results confirm that MOP and DOP have the potential to alleviate similar types of pain and that this effect could slightly differ between species.

Knock-In Mouse Models to Investigate the Functions of Opioid Receptors in vivo.

Due to their low expression levels, complex multi-pass transmembrane structure, and the current lack of highly specific antibodies, the assessment of endogenous G protein-coupled receptors (GPCRs) remains challenging. While most of the research regarding their functions was performed in heterologous systems overexpressing the receptor, recent advances in genetic engineering methods have allowed the generation of several unique mouse models. These animals proved to be useful to investigate numerous aspects underlying the physiological functions of GPCRs, including their endogenous expression, distribution, interactome, and trafficking processes. Given their significant pharmacological importance and central roles in the nervous system, opioid peptide receptors (OPr) are often referred to as prototypical receptors for the study of GPCR regulatory mechanisms. Although only a few GPCR knock-in mouse lines have thus far been generated, OPr are strikingly well represented with over 20 different knock-in models, more than half of which were developed within the last 5 years. In this review, we describe the arsenal of OPr (mu-, delta-, and kappa-opioid), as well as the opioid-related nociceptin/orphanin FQ (NOP) receptor knock-in mouse models that have been generated over the past years. We further highlight the invaluable contribution of such models to our understanding of the in vivo mechanisms underlying the regulation of OPr, which could be conceivably transposed to any other GPCR, as well as the limitations, future perspectives, and possibilities enabled by such tools.

Differential barcoding of opioid receptors trafficking.

Over the past several years, studies have highlighted the δ-opioid receptor (DOPr) as a promising therapeutic target for chronic pain management. While exhibiting milder undesired effects than most currently prescribed opioids, its specific agonists elicit effective analgesic responses in numerous animal models of chronic pain, including inflammatory, neuropathic, diabetic, and cancer-related pain. However, as compared with the extensively studied µ-opioid receptor, the molecular mechanisms governing its trafficking remain elusive. Recent advances have denoted several significant particularities in the regulation of DOPr intracellular routing, setting it apart from the other members of the opioid receptor family. Although they share high homology, each opioid receptor subtype displays specific amino acid patterns potentially involved in the regulation of its trafficking. These precise motifs or "barcodes" are selectively recognized by regulatory proteins and therefore dictate several aspects of the itinerary of a receptor, including its anterograde transport, internalization, recycling, and degradation. With a specific focus on the regulation of DOPr trafficking, this review will discuss previously reported, as well as potential novel trafficking barcodes within the opioid and nociceptin/orphanin FQ opioid peptide receptors, and their impact in determining distinct interactomes and physiological responses.

Identification of the interactome of the DP1 receptor for Prostaglandin D2: Regulation of DP1 receptor signaling and trafficking by IQGAP1.

BACKGROUND: Mechanisms governing localization, trafficking and signaling of G protein-coupled receptors (GPCRs) are critical in cell function. Protein-protein interactions are determinant in these processes. However, there are very little interacting proteins known to date for the DP1 receptor for prostaglandin D2. METHODS: We performed LC-MS/MS analyses of the DP1 receptor interactome in HEK293 cells. To functionally validate our LC-MS/MS data, we studied the implications of the interaction with the IQGAP1 scaffold protein in the trafficking and signaling of DP1. RESULTS: In addition to expected interacting proteins such as heterotrimeric G protein subunits, we identified proteins involved in signaling, trafficking, and folding localized in various cell compartments. Endogenous DP1-IQGAP1 co-immunoprecipitation was observed in colon cancer HT-29 cells. The interaction was augmented by DP1 agonist activation in HEK293 cells and GST-pulldown assays showed that IQGAP1 binds to intracellular loops 2 and 3 of DP1. Co-localization of the two proteins was observed by confocal microscopy at the cell periphery and in intracellular vesicles in the basal state. PGD2 treatment resulted in the redistribution of the DP1-IQGAP1 co-localization in the perinuclear vicinity. DP1 receptor internalization was promoted by overexpression of IQGAP1, while it was diminished by IQGAP1 knockdown with DsiRNAs. DP1-mediated ERK1/2 activation was augmented and sustained overtime by overexpression of IQGAP1 when compared to DP1 expressed alone. IQGAP1 knockdown decreased ERK1/2 activation by DP1 stimulation. Interestingly, ERK1/2 signaling by DP1 was increased when IQGAP2 was silenced, while it was impaired by IQGAP3 knockdown. CONCLUSIONS: Our findings define the putative DP1 interactome, a patho-physiologically important receptor, and validated the interaction with IQGAP1 in DP1 function. Our data also reveal that IQGAP proteins may differentially regulate GPCR signaling. GENERAL SIGNIFICANCE: The identified putative DP1-interacting proteins open multiple lines of research in DP1 and GPCR biology in various cell compartments.

In-depth NMR characterization of Rab4a structure, nucleotide exchange and hydrolysis kinetics reveals an atypical GTPase profile.

Rab4a is a small GTPase associated with endocytic compartments and a key regulator of early endosomes recycling. Gathering evidence indicates that its expression and activation are required for the development of metastases. Rab4a-intrinsic GTPase properties that control its activity, i.e. nucleotide exchange and hydrolysis rates, have not yet been thoroughly studied. The determination of these properties is of the utmost importance to understand its functions and contributions to tumorigenesis. Here, we used the constitutively active (Rab4aQ67L) and dominant negative (Rab4aS22N) mutants to characterize the thermodynamical and structural determinants of the interaction between Rab4a and GTP (GTPγS) as well as GDP. We report the first 1H, 13C, 15N backbone NMR assignments of a Rab GTPase family member with Rab4a in complex with GDP and GTPγS. We also provide a qualitative description of the extent of structural and dynamical changes caused by the Q67L and S22N mutations. Using a real-time NMR approach and the two aforementioned mutants as controls, we evaluated Rab4a intrinsic nucleotide exchange and hydrolysis rates. Compared to most small GTPases such as Ras, a rapid GTP exchange rate along with slow hydrolysis rate were observed. This suggests that, in a cellular context, Rab4a can self-activate and persist in an activated state in absence of regulatory mechanisms. This peculiar profile is uncommon among the Ras superfamily members, making Rab4a an atypical fast-cycling GTPase and may explain, at least in part, how it contributes to metastases.

In vivo mapping of a GPCR interactome using knockin mice.

With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

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.

L-type prostaglandin D synthase regulates the trafficking of the PGD2 DP1 receptor by interacting with the GTPase Rab4.

Accumulating evidence indicates that G protein-coupled receptors (GPCRs) interact with Rab GTPases during their intracellular trafficking. How GPCRs recruit and activate the Rabs is unclear. Here, we report that depletion of endogenous L-type prostaglandin D synthase (L-PGDS) in HeLa cells inhibited recycling of the prostaglandin D2 (PGD2) DP1 receptor (DP1) to the cell surface after agonist-induced internalization and that L-PGDS overexpression had the opposite effect. Depletion of endogenous Rab4 prevented l-PGDS-mediated recycling of DP1, and l-PGDS depletion inhibited Rab4-dependent recycling of DP1, indicating that both proteins are mutually involved in this pathway. DP1 stimulation promoted its interaction through its intracellular C terminus with Rab4, which was increased by l-PGDS. Confocal microscopy revealed that DP1 activation induces l-PGDS/Rab4 co-localization. l-PGDS/Rab4 and DP1/Rab4 co-immunoprecipitation levels were increased by DP1 agonist treatment. Pulldown assays with purified GST-l-PGDS and His6-Rab4 indicated that both proteins interact directly. l-PGDS interacted preferentially with the inactive, GDP-locked Rab4S22N variant rather than with WT Rab4 or with constitutively active Rab4Q67L proteins. Overexpression and depletion experiments disclosed that l-PGDS partakes in Rab4 activation following DP1 stimulation. Experiments with deletion mutants and synthetic peptides revealed that amino acids 85-92 in l-PGDS are involved in its interaction with Rab4 and in its effect on DP1 recycling. Of note, GTPγS loading and time-resolved FRET assays with purified proteins suggested that l-PGDS enhances GDP-GTP exchange on Rab4. Our results reveal how l-PGDS, which produces the agonist for DP1, regulates DP1 recycling by participating in Rab4 recruitment and activation.

MURC/CAVIN-4 facilitates store-operated calcium entry in neonatal cardiomyocytes.

Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca2+ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca2+ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies.

Monitoring the Aggregation of GPCRs by Fluorescence Microscopy.

G protein-coupled receptors (GPCRs) contain highly hydrophobic domains that are subject to aggregation when exposed to the crowded environment of the cytoplasm. Many events can lead to protein aggregation such as mutations, endoplasmic reticulum (ER) stress, and misfolding. These processes have been widely known to impact GPCR folding, maturation, and localization. Protein aggregates are transported toward the microtubule-organizing center via dynein to form a large juxta-nuclear structure called the aggresome, and in due course, are then targeted for degradation. Here, we describe a method to study aggregation of GPCRs by fluorescence microscopy.

Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents.

Genetically-modified animal models have significantly increased our understanding of the complex central nervous system circuits. Among these models, inducible transgenic mice whose specific gene expression can be modulated through a Cre recombinase/LoxP system are useful to study the role of specific peptides and proteins in a given population of cells. In the present study, we describe an efficient approach to selectively deliver a Cre-GFP to dorsal root ganglia (DRG) neurons. First, mice of different ages were injected in both hindpaws with a recombinant adeno-associated virus (rAAV2/9-CBA-Cre-GFP). Using this route of injection in mice at 5 days of age, we report that approximately 20% of all DRG neurons express GFP, 6 to 8 weeks after the infection. The level of infection was reduced by 50% when the virus was administered at 2 weeks of age. Additionally, the virus-mediated delivery of the Cre-GFP was also investigated via the intrathecal route. When injected intrathecally, the rAAV2/9-CBA-Cre-GFP virus infected a much higher proportion of DRG neurons than the intraplantar injection, with up to 51.6% of infected lumbar DRG neurons. Noteworthy, both routes of injection predominantly transduced DRG neurons over spinal and brain neurons.

Hypoxia-induced mobilization of NHE6 to the plasma membrane triggers endosome hyperacidification and chemoresistance.

The pH-dependent partitioning of chemotherapeutic drugs is a fundamental yet understudied drug distribution mechanism that may underlie the low success rates of current approaches to counter multidrug resistance (MDR). This mechanism is influenced by the hypoxic tumour microenvironment and results in selective trapping of weakly basic drugs into acidified compartments such as the extracellular environment. Here we report that hypoxia not only leads to acidification of the tumour microenvironment but also induces endosome hyperacidification. The acidity of the vesicular lumen, together with the alkaline pH of the cytoplasm, gives rise to a strong intracellular pH gradient that drives intravesicular drug trapping and chemoresistance. Endosome hyperacidification is due to the relocalization of the Na+/H+ exchanger isoform 6 (NHE6) from endosomes to the plasma membrane, an event that involves binding of NHE6 to the activated protein kinase C-receptor for activated C kinase 1 complex. These findings reveal a novel mechanism of hypoxia-induced MDR that involves the aberrant intracellular distribution of NHE6.

Involvement of the coatomer protein complex I in the intracellular traffic of the delta opioid receptor.

The delta opioid receptor (DOPr) is known to be mainly expressed in intracellular compartments. It remains unknown why DOPr is barely exported to the cell surface, but it seems that a substantial proportion of the immature receptor is trapped within the endoplasmic reticulum (ER) and the Golgi network. In the present study, we performed LC-MS/MS analysis to identify putative protein partners involved in the retention of DOPr. Analysis of the proteins co-immunoprecipitating with Flag-DOPr in transfected HEK293 cells revealed the presence of numerous subunits of the coatomer protein complex I (COPI), a vesicle-coating complex involved in recycling resident proteins from the Golgi back to the ER. Further analysis of the amino acid sequence of DOPr identified multiple consensus di-lysine and di-arginine motifs within the intracellular segments of DOPr. Using cell-surface ELISA and GST pulldown assays, we showed that DOPr interacts with COPI through its intracellular loops 2 and 3 (ICL2 and ICL3, respectively) and that the mutation of the K164AK166 (ICL2) or K250EK252 (ICL3) putative COPI binding sites increased the cell-surface expression of DOPr in transfected cells. Altogether, our results indicate that COPI is a binding partner of DOPr and provide a putative mechanism to explain why DOPr is highly retained inside the cells.

Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex.

Mechanisms that prevent aggregation and promote folding of nascent G protein-coupled receptors (GPCRs) remain poorly understood. We identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the ß-isoform of thromboxane A2 receptor (TPß) by yeast two-hybrid screening. CCT7 coimmunoprecipitated with overexpressed TPß and ß2-adrenergic receptor (ß2AR) in HEK 293 cells, but also with endogenous ß2AR. CCT7 depletion by small interfering RNA reduced total and cell-surface expression of both receptors and caused redistribution of the receptors to juxtanuclear aggresomes, significantly more so for TPß than ß2AR. Interestingly, Hsp90 coimmunoprecipitated with ß2AR but virtually not with TPß, indicating that nascent GPCRs can adopt alternative folding pathways. In vitro pull-down assays showed that both receptors can interact directly with CCT7 through their third intracellular loops and C-termini. We demonstrate that Trp334 in the TPß C-terminus is critical for the CCT7 interaction and plays an important role in TPß maturation and cell-surface expression. Of note, introducing a tryptophan in the corresponding position of the TPα isoform confers the CCT7-binding and maturation properties of TPß. We show that an interaction with a subunit of the CCT/TCP-1 ring complex (TRiC) chaperonin complex is involved in regulating aggregation of nascent GPCRs and in promoting their proper maturation and expression.

Thromboxane A2 receptor (TBXA2R) is a potent survival factor for triple negative breast cancers (TNBCs).

Triple Negative Breast Cancer (TNBC) is defined by the lack of ERα, PR expression and HER2 overexpression and is the breast cancer subtype with the poorest clinical outcomes. Our aim was to identify genes driving TNBC proliferation and/or survival which could represent novel therapeutic targets.We performed microarray profiling of primary TNBCs and generated differential genelists based on clinical outcomes following the chemotherapy regimen FEC (5-Fluorouracil/Epirubicin/Cyclophosphamide -'good' outcome no relapse > 3 years; 'poor' outcome relapse < 3 years). Elevated expression of thromboxane A2 receptor (TBXA2R) was observed in 'good' outcome TNBCs. TBXA2R expression was higher specifically in TNBC cell lines and TBXA2R knockdowns consistently showed dramatic cell killing in TNBC cells. TBXA2R mRNA and promoter activities were up-regulated following BRCA1 knockdown, with c-Myc being required for BRCA1-mediated transcriptional repression.We demonstrated that TBXA2R enhanced TNBC cell migration, invasion and activated Rho signalling, phenotypes which could be reversed using Rho-associated Kinase (ROCK) inhibitors. TBXA2R also protected TNBC cells from DNA damage by negatively regulating reactive oxygen species levels. In summary, TBXA2R is a novel breast cancer-associated gene required for the survival and migratory behaviour of a subset of TNBCs and could provide opportunities to develop novel, more effective treatments.

Characterization of the interaction between the prostaglandin D2 DP1 receptor and the intracellular L-prostaglandin D synthase.

Identification of G protein-coupled receptor (GPCR)-interacting proteins is an intense subject of current research. However, confirmation and characterization of identified interactions can be difficult with GPCRs, especially at the endogenous level. Here, we describe how we characterized the interaction between the prostaglandin D2 DP1 receptor and the intracellular L-type prostaglandin D synthase by in vitro pull-down assays using purified recombinant GST- and His-tagged proteins, by co-immunoprecipitation of overexpressed Flag- and HA-tagged proteins, and by co-immunoprecipitation of endogenous proteins.

New insights in the regulation of Rab GTPases by G protein-coupled receptors.

Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, forming the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Growing evidence suggests that mutations, aberrant expression, and altered post-translational modifications of Rab GTPases are associated with human diseases. However, their regulatory mechanisms and how they are connected to cargo proteins are still poorly understood. Accumulating data indicate that G protein-coupled receptors (GPCRs) directly associate with Rab GTPases and that these interactions dictate receptor trafficking. Yet, it remained unclear whether the receptors could regulate the targeting and activity of Rab GTPases in various cell compartments. It is only in recent years that experimental studies showed that GPCR signaling and interaction with Rab-associated regulatory proteins modulate the localization and activity of Rab GTPases. This research is revealing novel regulatory mechanisms of these small GTPases and should contribute to the progress in effective drug development. Recently published in the Journal of Cell Science, Lachance et al. present a novel role for ubiquitylation of Rab11a by a ß2AR/HACE1 complex in regulating Rab11a activity and ß2AR trafficking.

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.