DOI: 10.5061/dryad.0cfxpnwcq
title: Data from: In vitro signaling properties of cannabinoid and orexin receptors: how orexin receptors influence cannabinoid receptor-mediated signaling

description: The co-expression of different types of G protein-coupled receptors
 (GPCRs) in the same cells can have implications on receptor signaling and
 receptor cross-talk, potentially altering the apparent potency or efficacy
 of ligands targeting each receptor. The endocannabinoid and orexinergic
 systems, consisting of class A GPCRs and their endogenous ligands, are
 highly complex and regulate processes such as appetite, sleep,
 nociception, and energy homeostasis. The shared anatomical distribution of
 cannabinoid and orexin receptors in various regions of the central nervous
 system (CNS), coupled with data from previous studies exploring physical
 and functional interactions between these receptors, suggests that the
 endocannabinoid and orexinergic systems engage in crosstalk. In this
 study, we explored how orexin receptors (OX1, OX2) altered the in vitro
 signaling of cannabinoid receptors (CB1, CB2) in Chinese hamster ovary
 (CHO)-K1 cells by quantifying cyclic adenosine monophosphate (cAMP)
 inhibition and βarrestin2 recruitment. Our results suggest that orexin
 receptors alter agonist-dependent signaling at the cannabinoid receptors
 by enhancing cannabinoid receptor-mediated cAMP inhibition while
 increasing or decreasing cannabinoid receptor-mediated βarrestin2
 recruitment. These initial results are important for understanding the
 effects associated with cannabinoid ligands and may provide novel insights
 for therapeutics targeting physiological processes modulated by both
 systems.
description: # Data from: In vitro signaling properties of cannabinoid and orexin
 receptors: how orexin receptors influence cannabinoid receptor-mediated
 signaling
 [https://doi.org/10.5061/dryad.0cfxpnwcq](https://doi.org/10.5061/dryad.0cfxpnwcq) ## Description of the data and file structure File name: 2024-10-03_RawData_Manuscript_PharmacolResPersp_update.xlsx This Dryad submission includes an Excel file containing raw data and analyses related to the linked manuscript. Data are from: (1) Fluorescent microscopy assays that have been analyzed for the number of nuclei (Hoescht staining) and the number of green fluorescent protein (GFP)-positive cells where either the orexin 1 or orexin 2 receptor is tagged with GFP.  (2) cAMP inhibition assays that have been analyzed for cAMP inhibition following treatment of Chinese hamster ovary (CHO)- K1 HitHunter (DiscoveRx) cells stably expressing either the type 1 or type 2 cannabinoid receptor (CB1R, CB2R) and transfected with either orexin 1 or 2 (OX1, OX2) receptors treated with either no treatment (i.e., untreated), vehicle (10% dimethylsulfoxide [DMSO] in phosphate-buffered saline [PBS] containing 10 uM forskolin), or 0.1 nM - 10 uM CP55,940 dissolved in vehicle. These data were also analyzed for concentration-response relationships using nonlinear regression to estimate potency and efficacy. (3) b-arrestin2 recruitment assays that have been analyzed for b-arrestin2 recruitment following treatment of CHO-K1 PathHunter (DiscoveRx) cells stably expressing either CB1R or CB2R and transfected with either OX1 or OX2, treated with either no treatment (i.e., untreated), vehicle (10% DMSO in PBS), or 0.1 nM - 10 μM CP55, 940 dissolved in vehicle. These data were also analyzed for concentration-response relationships using nonlinear regression to estimate potency and efficacy. ### Description of the data and file structure Data are organized in an Excel file (file name: 2024-10-03_RawData_Manuscript_PharmacolResPersp.xlsx) such that each tab in the Excel file directly corresponds to a figure or figure panel appearing in the main manuscript data files. Excel file tabs are named according to the figure they correspond with. Separate tabs within the Excel files are also dedicated to analyses (e.g., nonlinear regression or analysis of variance). All such analyses have been copied/pasted from their original source files (GraphPad Prism version 9.0) into these Excel files for ease of accessibility. Specific notes - also present within each Excel file tab - to help guide users of the data are as follows: **1) Tab Fig. 1:** **Nuclei and GFP staining** a) This tab contains all data relating to Figure 1 of our manuscript. The percentage is calculated as GFP-positive cells divided by positive nuclei staining cells multiplied by 100. Total cell counts and their corresponding standard errors of the mean (SEM) are shown in column H. GFP-positive cells and their corresponding SEM are shown in column I. **2) Tab cAMP Fig 2A 3A 4A 5A:** **Baseline cAMP data** a) This tab contains all data relating to figure panels 2A, 3A, 4A, and 5A. b) Toward the top of the tab Excel rows 1-76 provide raw data shown in technical triplicate (and corresponding date) for each experiment in both the untreated (i.e., no treatment) and vehicle treated conditions (e.g., rows 2-6); followed by the mean of those triplicate values (e.g., rows 8-12); followed by the fold change in cAMP inhibition with the untreated condition set to '1'. The amount of OX1 or OX2 plasmid transfected, in ug, is shown in column 'A'. The experimental condition (e.g., CB1R-OX1R) is shown in the upper left of each section. Note that in the '0' (i.e., non-transfected) cells, CB1R or CB2R data for untreated and vehicle were combined to yield n=8, whereas all other conditions had n=4. c) Toward the bottom of this Excel tab, rows &gt;76 provide two-way ANOVA results copied/pasted from GraphPad Prism 9.0. Experimental conditions are demarked with yellow highlighting. **3) Tab cAMP Fig 2B 3B 4B 5B:** **cAMP concentration-response data** a) This tab contains all fold transform data for cAMP inhibition experiments. The data presented are the fold change in cAMP inhibition with the baseline condition constrained to '1' as described in the Methods of our paper. The amount of OX1 or OX2 plasmid transfected, in ug, is shown above each grouping of data. The experimental condition (e.g., CB1R-OX1R) is shown in the upper left of each section. Note that in the '0' (i.e., non-transfected) cells, CB1R or CB2R data were n=8, whereas all other conditions had n=4. **4) Tab barr2 Fig 2C 3C 4C 5C:** **Baseline B-arrestin2 recruitment data** a) This tab contains all data relating to figure panels 2C, 3C, 4C, and 5C. b) Toward the top of the tab Excel rows 1-76 provide raw data shown in technical triplicate (and corresponding date) for each experiment in both the untreated (i.e., no treatment) and vehicle treated conditions (e.g., rows 2-6); followed by the mean of those triplicate values (e.g., rows 8-12); followed by the fold change in B-arrestin2 recruitment with the untreated condition set to '1'. The amount of OX1 or OX2 plasmid transfected, in ug, is shown in column 'A'. The experimental condition (e.g., CB1R-OX1R) is shown in the upper left of each section. Note that in the '0' (i.e., non-transfected) cells, CB1R or CB2R data for untreated and vehicle were combined to yield n=8, whereas all other conditions had n=4. c) Toward the bottom of this Excel tab, rows &gt;76 provide two-way ANOVA results copied/pasted from GraphPad Prism 9.0. Experimental conditions are demarked with yellow highlighting. **5) Tab barr2 Fig 2D 3D 4D 5D:** **B-arrestin2 concentration-response data** a) This tab contains all fold transform data for B-arrestin2 experiments. The data presented are the fold change in B-arrestin2 recruitment with the baseline condition constrained to '1' as described in the Methods of our paper. The amount of OX1 or OX2 plasmid transfected, in ug, is shown above each grouping of data. The experimental condition (e.g., CB1R-OX1R) is shown in the upper left of each section. Note that in the '0' (i.e., non-transfected) cells, CB1R or CB2R data were n=8, whereas all other conditions had n=4. **6) Tab Fig. 2B Table 1** a) This tab contains all analyses related to nonlinear regression of CB1R-OX1R cAMP inhibition assays in Fig. 2 and Table 1. b) In this Tab, the model used is highlighted in yellow or green. For some treatments, the four-parameter nonlinear regression produced a poor fit, and a three-parameter model was used instead (as described in our Manuscript). As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. For example, "Top" (i.e., Emax) is calculated as the mean of the highest observed response for the indicated compound. c) Tabulated Emax and LogEC50 values are provided in rows 26-31. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 33. **7) Tab Fig. 2D Table 1** a) This tab contains all analyses related to nonlinear regression of CB1R-OX1R barrestin2 recruitment assays in Fig. 2 and Table 1. b) In this Tab, the model used is highlighted in yellow or green. As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. For example, "Top" (i.e., Emax) is calculated as the mean of the highest observed response for the indicated compound. This is specifically shown, for example, with green and red highlighting in rows 28 and 32 of this tab. c) Tabulated Emax and LogEC50 values are provided in rows 26-31. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 33. **8) Tab Fig. 3B Table 2** a) This tab contains all analyses related to nonlinear regression of CB1R-OX2R cAMP inhibition assays in Fig. 3 and Table 2. b) In this Tab, the model used is highlighted in yellow. As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. For example, "Top" (i.e., Emax) is calculated as the mean of the highest observed response for the indicated compound. For example, LogEC50 could not be estimated for these data in the 0.25 and 0.50 μg conditions. This is specifically shown, for example, with red highlighting in rows 31-33 of this tab. c) Tabulated Emax and LogEC50 values are provided in rows 26-32. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 34. Where ANOVA was not possible, this was highlighted in red. **9) Tab Fig. 3D Table 2** a) This tab contains all analyses related to nonlinear regression of CB1R-OX2R barrestin2 recruitment assays in Fig. 3 and Table 2. b) In this Tab, the model used is highlighted in yellow. As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. For example, "Top" (i.e., Emax) is calculated as the mean of the highest observed response for the indicated compound. This is specifically shown, for example, with green and red highlighting in rows 29-33of this tab. c) Tabulated Emax and LogEC50 values are provided in rows 27-33. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 34. Where ANOVA was not possible, this was highlighted in red. **10) Tab Fig. 4B Table 3** a) This tab contains all analyses related to nonlinear regression of CB2R-OX1R cAMP inhibition assays in Fig. 4 and Table 3. b) In this Tab, the model used is highlighted in yellow or green. For some treatments, the four-parameter nonlinear regression produced a poor fit, and a three-parameter model was used instead (as described in our Manuscript). As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. For example, "Top" (i.e., Emax) and "Bottom" (i.e., Emin) were not converged due to poor fit (red highlighting in rows 30-33 of this tab_. c) Tabulated Emax and LogEC50 values are provided in rows 26-31. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 34. Where ANOVA was not possible, this was highlighted in red. **11) Tab Fig. 4D Table 3** a) This tab contains all analyses related to nonlinear regression of CB2R-OX1R barrestin2 recruitment assays in Fig. 4 and Table 3. b) In this Tab, the model used is highlighted in yellow. As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. No examples of this occurring are present within this tab. c) Tabulated Emax and LogEC50 values are provided in rows 25-30. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 32. **12) Tab Fig. 5B Table 4** a) This tab contains all analyses related to nonlinear regression of CB2R-OX2R cAMP inhibition assays in Fig. 5 and Table 4. b) In this Tab, the model used is highlighted in yellow or green. For some treatments, the four-parameter nonlinear regression produced a poor fit, and a three-parameter model was used instead (as described in our Manuscript). As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. No examples of this occurring are present within this tab. c) Tabulated Emax and LogEC50 values are provided in rows 27-32. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 34. **13) Tab Fig. 5D Table 4** a) This tab contains all analyses related to nonlinear regression of CB2R-OX2R B-arrestin2 recruitment assays in Fig. 5 and Table 4. b) In this Tab, the model used is highlighted in yellow or green. For some treatments, the four-parameter nonlinear regression produced a poor fit, and a three-parameter model was used instead (as described in our Manuscript). As noted in the Excel file, GraphPad Prism output of "???" or "(Very wide)" indicates that nonlinear regression analyses were unable to estimate a 95% confidence interval for the specified variable. Likewise, if GraphPad Prism is unable to output a best fit value, the field is populated with the result "Unstable". In these cases, values are calculated based on group means directly from data. No examples of this occurring are present within this tab. c) Tabulated Emax and LogEC50 values are provided in rows 27-32. d) ANOVA and post-hoc analyses for Emax and LogEC50 values are provided below row 34. ### Files and variables #### File: 2024-10-03_RawData_Manuscript_PharmacolResPersp.xlsx **Description:** In this Excel file, data are organized such that each tab in the Excel file directly corresponds to a figure or figure panel appearing in the main manuscript data files. Excel file tabs are named according to the figure they correspond with. Separate tabs within the Excel files are also dedicated to analyses (e.g., nonlinear regression or analysis of variance). All such analyses have been copied/pasted from their original source files (GraphPad Prism version 9.0) into these Excel files for ease of accessibility. ##### Variables * Tab 1, "Fig. 1": * \#Nuclei = number of Hoescht-stained positive cells; # GFP-+ cells = number of GFP-stained cells; Percentage = (#GFP-+ cells/#Nuclei)*100%; column H mean of replicates for # nuclei with standard error of the mean (SEM) shown below; column I mean of percentage with SEM shown below * Tab 2, "cAMP Fig 2A, 3A, 4A, 5A": measured luminescence of cAMP inhibition assay (e.g., rows 2-6); mean of technical replicates for luminescence for cAMP inhibition assay (e.g., rows 8-12); mean fold of technical replicates of cAMP inhibition assay (e.g., rows 14-18); amount of orexin receptor plasmid transfected, in micrograms (ug) shown in column A. * Tab 3, "cAMP Fig 2B, 3B, 4B, 5B": measured mean fold of technical replicates of cAMP inhibition assay (e.g., rows 4-9); amount of orexin receptor plasmid transfected, in micrograms (ug) (e.g., row 3); Log[CP55,940] is the logarithm of concentration for compound CP55,940 in units molar (M) (e.g., -5 = 10^-5 = 10 micromolar). * Tab 4, "barr2 Fig 2C, 3C, 4C, 5C": measured luminescence of barrestin2 recruitment assay (e.g., rows 2-6); mean of technical replicates for luminescence for barrestin2 recruitment assay (e.g., rows 8-12); mean fold of technical replicates of barrestin2 recruitment assay (e.g., rows 14-18); amount of orexin receptor plasmid transfected, in micrograms (ug) shown in column A. * Tab 5, "barr2 Fig 2D, 3D, 4D, 5D": measured mean fold of technical replicates of barrestin2 recruitment assay (e.g., rows 4-9); amount of orexin receptor plasmid transfected, in micrograms (ug) (e.g., row 3); Log[CP55,940] is the logarithm of concentration for compound CP55,940 in units molar (M) (e.g., -5 = 10^-5 = 10 micromolar). * Tabs 6-13: Non-linear regression analyses and analysis of variance (ANOVA) for corresponding data. Bottom = bottom of concentration-response curve (CRC) in units fold; Top = top of CRC in units fold; LogEC50 = log of effective concentration 50 (i.e., EC50) concentration to produce 50% maximal effect in units log molar; HillSlope = slope of the linear portion of the CRC, unitless; EC50 = antilog of LogEC50 in units molar; Span = distance in units fold from Bottom to Top; 95% CI span of error surrounding each parameter estimate described above; Goodness of Fit = parameters showing residual fit to the nonlinear regression that are unitless (degrees of freedom, R squared, Sum of Squares) or in units Fold (Sy.x). Nonlinear regression estimated parameters (e.g., tab "Fig. 2B Table 1") show the estimated Emax, Emin (fold); LogEC50 (logarithm units molar); HillSlope (unitless); and EC50 (molar). The concentration of orexin transfected is shown in units of micrograms (μg). **Abbreviations** * All tabs: cAMP, cyclic adenosine monophosphate; CB1R, type 1 cannabinoid receptor; CB2R, type 2 cannabinoid receptor; OX1R, orexin receptor 1; OX2R, orexin receptor 2; barr2, b-arrestin2; BLANK, no transfection; ANOVA, analysis of variance; LogEC50 = log of effective concentration 50 (i.e., EC50) concentration to produce 50% maximal effect in units log molar; CI, confidence interval. **Missing Values** * Missing values or values that cannot be estimated are shown with "???" as described in the ReadMe and/or are highlighted in green and red in the Excel file, with explanations provided with the Excel file and the ReadMe. ## Code/software Microsoft Excel (.xlsx), LibreOffice ## Access information Other publicly accessible locations of the data: * Data related to this manuscript are also presented in the linked published paper and its corresponding supplementary information. Data was derived from the following sources: * Original experiments only