Natasha L. Grimsey

Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal!

The study of endogenous cannabinoid CB1 receptor proteins in neuronal tissues and cells relies on the availability of highly specific antibodies. We have tested the ability of a series of CB1 antibodies to detect endogenous receptors in brain as well as hemagglutinin (HA)-tagged receptors transfected into HEK-293 cells using a combination of immunological methods. An initial comparison of several CB1 antibodies in mouse brain revealed substantial differences in staining pattern to ligand binding by autoradiography. Antibodies were then tested immunocytochemically against HEK cells expressing HA-tagged rat and human CB1 receptors. None of the commercial antibodies tested were able to detect the receptor in this context. All antibodies were then screened by Western blotting using lysates from the HEK cells and rodent brain homogenates. Again, none of the commercially available antibodies detected proteins of the correct molecular weight in transfected cell lines or brain homogenates, although all recognized multiple proteins in brain tissues. We conclude that the commercially available antibodies we tested failed to detect CB1 receptors abundantly expressed in HEK cells or native receptors in brain slices or homogenates. As such, comprehensive validation of the specificity of these CB1 antibodies for a particular application is essential before use.

Real‐time characterization of cannabinoid receptor 1 (CB1) allosteric modulators reveals novel mechanism of action

The cannabinoid receptor type 1 (CB1) has an allosteric binding site. The drugs ORG27569 {5‐chloro‐3‐ethyl‐N‐[2‐[4‐(1‐piperidinyl)phenyl]ethyl]‐1H‐indole‐2‐carboxamide} and PSNCBAM‐1 {1‐(4‐chlorophenyl)‐3‐[3‐(6‐pyrrolidin‐1‐ylpyridin‐2‐yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(−)‐cis‐3‐[2‐hydroxy‐4‐(1,1‐dimethylheptyl)phenyl]‐trans‐4‐(3‐hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non‐competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1, we examined real‐time signalling and trafficking responses of the receptor in the presence of allosteric modulators.

Cannabinoid receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway

Cannabinoid receptor 2 (CB2) is a GPCR highly expressed on the surface of cells of the immune system, supporting its role in immunomodulation. This study has investigated the trafficking properties of this receptor when stably expressed by HEK-293 cells. As previously reported, cell surface CB2 rapidly internalized upon exposure to agonist. Direct evidence of CB2 recycling was observed upon competitive removal of the stimulating agonist by inverse agonist. CB2 also underwent slow constitutive internalization when agonist was absent and was up-regulated in the presence of inverse agonist. Co-expression of CB2 and dominant negative Rab5 resulted in a significantly reduced capacity for receptors to internalize with no effect on recycling of the internalized receptors. Conversely, co-expression with dominant negative Rab11 did not alter the ability of CB2 to internalize but did impair their ability to return to the cell surface. Co-expression of wild-type, dominant negative or constitutively active Rab4 with CB2 did not alter basal surface expression, extent of internalization, or extent of recycling. These results suggest that Rab5 is involved in CB2 endocytosis and that internalized receptors are recycled via a Rab11 associated pathway rather than the rapid Rab4 associated pathway. This report provides the first comprehensive description of CB2 internalization and recycling to date.

GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine

The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly). Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP) and phosphorylation of extracellular signal-regulated kinase (ERK), in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R), were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence) and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine). This A108N mutant did cause an increase in surface receptor expression (which may argue for reduced constitutive activity), but no ligand-mediated effects were detected. Two glioblastoma multiforme cell lines (which endogenously express GPR18) were assayed for NAGly-induced pERK phosphorylation, with negative results. Despite a lack of ligand-mediated responses in all assays, the constitutive trafficking of GPR18 remains an interesting facet of receptor function and will have consequences for understanding the role of GPR18 in physiology.

A NOVEL HIGH‐THROUGHPUT ASSAY FOR THE QUANTITATIVE ASSESSMENT OF RECEPTOR TRAFFICKING

1 Receptor transport between intracellular compartments has important consequences for receptor function and is an exciting area of current study. Existing methods for studying receptor trafficking often require labour‐intensive techniques or are difficult to quantify reliably. We report a novel high‐throughput method that uses automated imaging and analysis tools to accurately quantify cannabinoid CB1 receptor trafficking. 2 Haemagglutinin (HA)‐tagged CB1 was stably expressed in HEK‐293 cells and cell surface or total receptors were detected immunocytochemically. Images of receptor and nuclear staining were acquired with an automated fluorescent microscope (Discovery‐1; Molecular Devices, Sunnyvale, CA, USA) and quantified at high throughput with MetaMorph (Molecular Devices) software. The ‘Granularity’ assay measured internalization by counting receptor clusters that appear during receptor endocytosis, a well‐established approach. Our assay, referred to as ‘Total Grey Value per Cell’ (TGVC), measures the total fluorescence above background, normalized to cell count. 3 Incubation with the cannabinoid agonist HU‐210 (100 nmol/L) resulted in rapid CB1 internalization, reaching a maximum within 20 min. Whether quantified by Granularity or TGVC, the time‐course of endocytosis could be modelled with exponentially derived curves and with similar half‐lives. We demonstrate the sensitivity of our TGVC method by measuring the concentration dependence of CB1 internalization and its versatility by measuring downregulation following chronic agonist exposure, whereby total CB1 was reduced to approximately 55% of basal after 3 h. 4 The TGVC quantification method described is efficient, accurate and versatile and is likely to provide a valuable tool in receptor trafficking studies.

Identification of N‐arachidonoyl dopamine as a highly biased ligand at cannabinoid CB1 receptors

N‐arachidonyl dopamine (NADA) has been identified as a putative endocannabinoid, but there is little information about which signalling pathways it activates. The purpose of this study was to identify the signalling pathways activated by NADA in vitro.

Sensitive and Accurate Quantification of Human Leukocyte Migration Using High-Content Discovery-1 Imaging System and ATPlite Assay.

Migration is a fundamental aspect of leukocyte behavior and represents a significant therapeutic target clinically. However, most migration assays used in research are relatively low throughput and not easily compatible with rapid analysis or high-throughput screening (HTS) protocols required for drug screening assays. We therefore investigated the quantification of the migration of human leukocytes using the Molecular Devices high-content Discovery-1 platform or PerkinElmer ATPlite assay compared to manual counting. We have conducted extensive assay validation, investigating the detection limits, sensitivity, and precision of each method to count human leukocytes. Leukocyte migration assays were conducted using 96-well HTS-Transwell plates and the potent chemokine stromal cell–derived factor-1 (SDF-1). We reveal that the Discovery-1 and ATPlite methods developed here provide useful approaches to quantify leukocyte migration in an HTS manner with high levels of detection, sensitivity, and precision.

Gαs signalling of the CB1 receptor and the influence of receptor number

CB1 receptor signalling is canonically mediated through inhibitory Gαi proteins, but occurs through other G proteins under some circumstances, Gαs being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gαi blockade, CB1/D2 receptor co‐stimulation, CB1 agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB1 receptor signalling.

Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model

G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags.

Development of selective, fluorescent cannabinoid type 2 receptor ligands based on a 1,8-naphthyridin-2-(1H)-one-3-carboxamide scaffold

Cannabinoid type 2 (CB2) receptor has been implicated in several diseases and conditions, however no CB2 receptor selective drugs have made it to market. The aim of this study was to develop fluorescent ligands as CB2 receptor tools, to enable an increased understanding of CB2 receptor expression and signalling and thereby accelerate drug discovery. Fluorescent ligands have been successfully developed for other receptors, however none with adequate subtype selectivity or imaging properties have been reported for CB2 receptor. A series of 1,8-naphthyridin-2-(1H)-one-3-carboxamides with linkers and fluorophores appended in the N1 and C3-positions were developed. Molecular modelling indicated the C3 cis-cyclohexanol-linked compounds directed the linker out of the CB2 receptor between transmembrane helices 1 and 7. Herein we report fluorescent ligand 32 (hCB2 pK i = 6.33 ± 0.02) as one of the highest affinity, selective CB2 receptor fluorescent ligands reported. Despite 32 displaying poor specific labelling of CB2 receptor, the naphthyridine scaffold with this linker remains highly promising for future development of CB2 receptor tools.