Instrumentation development, improvement, simplification, and miniaturization: The multifunctional plate source for use in mass spectrometry.

In remembrance of Prof. Dr Przybylski, we are presenting a vision towards his beloved mass spectrometry (MS) and its far-reaching promises outside of the academic laboratory. Sub-atmospheric pressure (AP) ionization MS is well positioned to make a step-change in direct ionization, a concept that allows sublimation/evaporation ionization and mass analyses of volatile and nonvolatile molecules from clean or dirty samples, directly, accurately, sensitively, and in a straightforward manner that has the potential to expand the field of MS into unchartered application areas. Contrary to ambient ionization MS, ionization commences in the sub-AP region of the mass spectrometer, important for practical and safety reasons, and offers inter alia, simplicity, speed, sensitivity, and robustness directly from real-world samples without cleanup. The plate source concept, presented here, provides an easy to use, rapid, and direct sample introduction from AP into the sub-AP of a mass spectrometer. Utilizing sub-AP ionization MS based on the plate source concept, small to large molecules from various environments that would be deemed too dirty for some direct MS methods are demonstrated. The new source concept can be expanded to include multiple ionization methods using the same plate source "front end" without the need to vent the mass spectrometer between the different methods, thus allowing ionization of more compounds on the same mass spectrometer for which any one ionization method may be insufficient. Examples such as fentanyl, gamma-hydroxybutyric acid, clozapine, 1-propionyllysergic acid, hydrocodone angiotensin I and II, myoglobin, and carbonic anhydrase are included.

An overview of biological applications and fundamentals of new inlet and vacuum ionization technologies.

RATIONALE: The developments of new ionization technologies based on processes previously unknown to mass spectrometry (MS) have gained significant momentum. Herein we address the importance of understanding these unique ionization processes, demonstrate the new capabilities currently unmet by other methods, and outline their considerable analytical potential. METHODS: The inlet and vacuum ionization methods of solvent-assisted ionization (SAI), matrix-assisted ionization (MAI), and laserspray ionization can be used with commercial and dedicated ion sources producing ions from atmospheric or vacuum conditions for analyses of a variety of materials including drugs, lipids, and proteins introduced from well plates, pipet tips and plate surfaces with and without a laser using solid or solvent matrices. Mass spectrometers from various vendors are employed. RESULTS: Results are presented highlighting strengths relative to ionization methods of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization. We demonstrate the utility of multi-ionization platforms encompassing MAI, SAI, and ESI and enabling detection of what otherwise is missed, especially when directly analyzing mixtures. Unmatched robustness is achieved with dedicated vacuum MAI sources with mechanical introduction of the sample to the sub-atmospheric pressure (vacuum MAI). Simplicity and use of a wide array of matrices are attained using a conduit (inlet ionization), preferably heated, with sample introduction from atmospheric pressure. Tissue, whole blood, urine (including mouse, chicken, and human origin), bacteria strains and chemical on-probe reactions are analyzed directly and, especially in the case of vacuum ionization, without concern of carryover or instrument contamination. CONCLUSIONS: Examples are provided highlighting the exceptional analytical capabilities associated with the novel ionization processes in MS that reduce operational complexity while increasing speed and robustness, achieving mass spectra with low background for improved sensitivity, suggesting the potential of this simple ionization technology to drive MS into areas currently underserved, such as clinical and medical applications.

Discovery and characterization of two novel CB1 receptor splice variants with modified N-termini in mouse.

Numerous studies have been carried out in the mouse model, investigating the role of the cannabinoid receptor type 1 (CB1). However, mouse CB1 (mCB1) receptor differs from human CB1 (hCB1) receptor in 13 amino acid residues. Two splice variants, hCB1a and hCB1b, diverging in their amino-termini, have been reported to be unique for hCB1 and, via different signaling properties, contribute to CB1 receptor physiology and pathophysiology. We hypothesized that splice variants also exist for the mCB1 receptor and have different signaling properties. On murine hippocampal cDNA, we identified two novel mCB1 receptor splice variants generated by splicing of introns with 117 bp and 186 bp in the N-terminal domain, corresponding to deletions of 39 or 62 amino acids, respectively. The mRNAs for the splice variants mCB1a and mCB1b are expressed at low levels in different brain regions. Western blot analysis of protein extracts from stably transfected HEK293 cells indicates a strongly reduced glycosylation because of the absence of two glycosylation sites in mCB1b. On-cell western analysis in these stable lines revealed increased internalization of mCB1a and mCB1b upon stimulation with the agonist WIN55,212-2 as compared to mCB1. Results also point toward an increased affinity to SR141716 for mCB1a, as well as slightly enhanced inhibition of neurotransmission compared to mCB1. In mCB1b, agonist-induced MAPK phosphorylation was decreased compared to mCB1 and mCB1a. Identification of mouse CB1 receptor splice variants may help to explain differences found between human and mouse endocannabinoid systems and improve the understanding of CB1 receptor signaling and trafficking in different species.

Novel indole-based compounds that differentiate alkylindole-sensitive receptors from cannabinoid receptors and microtubules: Characterization of their activity on glioma cell migration.

Indole-based compounds, such as the alkyl-indole (AI) compound WIN55212-2, activate the cannabinoid receptors, CB1 and CB2, two well-characterized G protein-coupled receptors (GPCR). Reports indicate that several indole-based cannabinoid agonists, including WIN55212-2, lack selectivity and interact with at least two additional targets: AI-sensitive GPCRs and microtubules. Studying how indole-based compounds modulate the activity of these 4 targets has been difficult as selective chemical tools were not available. Here we report the pharmacological characterization of six newly-developed indole-based compounds (ST-11, ST-23, ST-25, ST-29, ST-47 and ST-48) that exhibit distinct binding affinities at AI-sensitive receptors, cannabinoid CB1 and CB2 receptors and the colchicine site of tubulin. Several compounds exhibit some level of selectivity for AI-sensitive receptors, including ST-11 that binds AI-sensitive receptors with a Kd of 52nM and appears to have a weaker affinity for the colchicine site of tubulin (Kd=3.2µM) and does not bind CB1/CB2 receptors. Leveraging these characteristics, we show that activation of AI-sensitive receptors with ST-11 inhibits both the basal and stimulated migration of the Delayed Brain Tumor (DBT) mouse glioma cell line. Our study describes a new series of indole-based compounds that enable the pharmacological and functional differentiation of alkylindole-sensitive receptors from cannabinoid receptors and microtubules.

A broad-based study on hyphenating new ionization technologies with MS/MS for PTMs and tissue characterization.

Matrix-assisted ionization (MAI) is a newly discovered method for converting compounds from the solid phase to gas-phase ions having charge states similar to electrospray ionization (ESI), but without the need for high-energy sources such as lasers or high voltage. Laserspray ionization (LSI) is a subset of MAI that uses a laser to provide high spatial resolution analyses, but the laser is not directly involved in the ionization process. These methods produce multiply-charged analyte ions that are useful for characterizing compounds directly from surfaces using advanced characterization technologies. Because the multiply-charged ions originate from charged matrix clusters, efficient desolvation of the matrix is a prerequisite. Here, we report on the utility of collision-induced dissociation (CID) and electron transfer dissociation (ETD) coupled to mass spectrometry using several MAI and LSI matrices for peptide and protein characterization employing mass spectrometers from two manufacturers. The information obtained is similar to that using ESI for most analyses and superior to matrix-assisted laser desorption/ionization (MALDI) as is shown for intact proteins and protein digests directly from mouse brain tissue sections. The ionization processes are soft so that posttranslational modification (e.g. phosphorylation) sites are readily determined. Instances where ETD or CID in conjunction with MAI failed are attributed to lack of desolvation of charged matrix:analyte particles.

Matrix-Assisted Ionization on a Portable Mass Spectrometer: Analysis Directly from Biological and Synthetic Materials.

Matrix-assisted ionization (MAI)-mass spectrometry (MS) eliminates the need for high voltage, a heat source, lasers, and compressed gases in the ionization process and uses minimal solvents in sample preparation, thus making MAI ideal for field-portable mass spectrometers. The broad applicability of MAI is demonstrated by simple, rapid, and robust positive and negative detection mode analyses of low and high mass compounds including some pesticides, dyes, drugs, lipids, and proteins (186 Da to 8.5 kDa) from various materials including urine, biological tissue sections, paper, and plant material on a low pumping capacity, single-quadrupole mass spectrometer. Different sample introduction methods are applicable, including the use of a pipet tip or glass melting point tube, allowing integration of sample preparation with sample introduction for increased analytical utility and ease of operation, even when sampling directly from surfaces.

Drug detection and quantification directly from tissue using novel ionization methods for mass spectrometry.

Solvent assisted ionization inlet (SAII) and matrix assisted ionization vacuum (MAIV) were used to quantify rapidly an antipsychotic drug, clozapine, directly from surfaces with minimal sample preparation. This simple surface analysis method based on SAII- and MAIV-mass spectrometry (MS) was developed to allow the detection of endogenous lipids, metabolites, and clozapine directly from sections of mouse brain tissue. A rapid surface assessment was achieved by SAII with the assistance of heat applied to the mass spectrometer inlet. MAIV provided an improved reproducibility without the need of a heated inlet. In addition, isotope dilution and standard addition were used without sample clean-up, and the results correlate well with liquid chromatography tandem MS using sample work-up. Using the simple surface methods, standard solutions containing clozapine and a deuterated internal standard (clozapine-d8) at different concentration ratios were used in the extraction and quantification of clozapine from brain tissue sections of a drug-treated mouse using different tissue thicknesses. The amount of clozapine extracted by these surface methods was independent of tissue thickness.

Cross-generational impact of a male murine pheromone 2-sec-butyl-4,5- dihydrothiazole in female mice.

The current understanding of the activity of mammalian pheromones is that endocrine and behavioural effects are limited to the exposed individuals. Here, we demonstrate that the nasal exposure of female mice to a male murine pheromone stimulates expansion of mammary glands, leading to prolonged nursing of pups. Subsequent behavioural testing of the pups from pheromone-exposed dams exhibited enhanced learning. Sialic acid components in the milk are known to be involved in brain development. We hypothesized that the offspring might have received more of this key nutrient that promotes brain development. The mRNA for polysialyltransferase, which produces polysialylated neural cell adhesion molecules related to brain development,was increased in the brain of offspring of pheromone-exposed dams at post-natal day 10, while it was not different at embryonic stages, indicating possible differential brain development during early post-natal life.

Molecular-interaction and signaling profiles of AM3677, a novel covalent agonist selective for the cannabinoid 1 receptor.

The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor's ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677's ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677's topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.

Transmission geometry laserspray ionization vacuum using an atmospheric pressure inlet.

This represents the first report of laserspray ionization vacuum (LSIV) with operation directly from atmospheric pressure for use in mass spectrometry. Two different types of electrospray ionization source inlets were converted to LSIV sources by equipping the entrance of the atmospheric pressure inlet aperture with a customized cone that is sealed with a removable glass plate holding the matrix/analyte sample. A laser aligned in transmission geometry (at 180° relative to the inlet) ablates the matrix/analyte sample deposited on the vacuum side of the glass slide. Laser ablation from vacuum requires lower inlet temperature relative to laser ablation at atmospheric pressure. However, higher inlet temperature is required for high-mass analytes, for example, α-chymotrypsinogen (25.6 kDa). Labile compounds such as gangliosides and cardiolipins are detected in the negative ion mode directly from mouse brain tissue as intact doubly deprotonated ions. Multiple charging enhances the ion mobility spectrometry separation of ions derived from complex tissue samples.

GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination.

The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior.

Laserspray ionization imaging of multiply charged ions using a commercial vacuum MALDI ion source.

This is the first report of imaging mass spectrometry (MS) from multiply charged ions at vacuum. Laserspray ionization (LSI) was recently extended to applications at vacuum producing electrospray ionization-like multiply charged ions directly from surfaces using a commercial intermediate pressure matrix-assisted laser desorption/ionization ion mobility spectrometry (IMS) MS instrument. Here, we developed a strategy to image multiply charged peptide ions. This is achieved by the use of 2-nitrophloroglucinol as matrix for spray deposition onto the tissue section and implementation of "soft" acquisition conditions including lower laser power and ion accelerating voltages similar to electrospray ionization-like conditions. Sufficient ion abundance is generated by the vacuum LSI method to employ IMS separation in imaging multiply charged ions obtained on a commercial mass spectrometer ion source without physical instrument modifications using the laser in the commercially available reflection geometry alignment. IMS gas-phase separation reduces the complexity of the ion signal from the tissue, especially for multiply charged relative to abundant singly charged ions from tissue lipids. We show examples of LSI tissue imaging from charge state +2 of three endogenous peptides consisting of between 1 and 16 amino acid residues from the acetylated N-terminal end of myelin basic protein: mass-to-charge (m/z) 795.81 (+2) molecular weight (MW) 1589.6, m/z 831.35 (+2) MW 1660.7, and m/z 917.40 (+2) MW 1832.8.

siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration.

BACKGROUND: Neurons are known to employ the endogenous cannabinoid system to communicate with other cells of the CNS. Endocannabioid signaling recruits microglia toward neurons by engaging cannabinoid CB2 and abnormal cannabidiol (Abn-CBD) receptors. The Abn-CBD receptor is a prominent atypical cannabinoid receptor that had been discriminated by means of various pharmacological and genetic tools but remained to be identified at the molecular level. We recently introduced N-arachidonoyl glycine (NAGly) signaling via GPR18 receptors as an important novel signaling mechanism in microglial-neuronal communication. NAGly is an endogenous, enzymatically oxygenated metabolite of the endocannabinoid N-arachidonoyl ethanolamide (AEA). Our recent studies support strongly two hypotheses; first that NAGly initiates directed microglial migration in the CNS through activation of GPR18, and second that GPR18 is the Abn-CBD receptor. Here we present siRNA knockdown data in further support of these hypotheses. FINDINGS: A GPR18-targetting siRNA pSUPER G418 GFP cDNA plasmid was created and transfected into BV-2 microglia. Successfully transfected GFP+ GPR18 siRNA BV-2 microglia displayed reduced GPR18 mRNA levels and immunocytochemical staining. Cell migration induced by 1 µM concentrations of NAGly, O-1602 and Abn-CBD were significantly attenuated in GFP+ cells. CONCLUSIONS: Our data provide definitive evidence that these compounds, characteristic of Abn-CBD receptor pharmacology, are acting via GPR18 in BV-2 microglia. A fuller understanding of the hitherto unidentified cannabinoid receptors such as GPR18; their molecular interactions with endogenous ligands; and how phytocannabinoids influence their signaling is vital if we are to comprehensively assess the function of the endogenous cannabinoid signaling system in human health and disease.

Localization and imaging of gangliosides in mouse brain tissue sections by laserspray ionization inlet.

A new ionization method for the analysis of fragile gangliosides without undesired fragmentation or salt adduction is presented. In laserspray ionization inlet (LSII), the matrix/analyte sample is ablated at atmospheric pressure, and ionization takes place in the ion transfer capillary of the mass spectrometer inlet by a process that is independent of a laser wavelength or voltage. The softness of LSII allows the identification of gangliosides up to GQ1 with negligible sialic acid loss. This is of importance to the field of MS imaging, as undesired fragmentation has made it difficult to accurately map the spatial distribution of fragile ganglioside lipids in tissue. Proof-of-principle structural characterization of endogenous gangliosides using MS(n) fragmentation of multiply charged negative ions on a LTQ Velos and subsequent imaging of the GD1 ganglioside is demonstrated. This is the first report of multiply charged negative ions using inlet ionization. We find that GD1 is detected at higher levels in the mouse cortex and hippocampus compared with the thalamus. In LSII with the laser aligned in transmission geometry relative to the inlet, images were obtained in approximately 60 min using an inexpensive nitrogen laser.

Functional selectivity in CB(2) cannabinoid receptor signaling and regulation: implications for the therapeutic potential of CB(2) ligands.

Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB(1) and CB(2) cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB(2) to different extents. Because CB(2) is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB(1) and CB(2) trafficking. Our most striking finding was that (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB(2) receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB(2) receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB(2) internalization. Despite these differences in internalization, both compounds activated CB(2) receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of ß-arrestin(2) to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB(2) receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB(2) ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB(2) receptors and suggest that different classes of CB(2) ligands may produce diverse physiological effects, emphasizing that each class needs to be separately evaluated for therapeutic efficacy.

Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis.

BACKGROUND: G protein coupled receptors (GPCRs) are one of the most widely studied gene superfamilies. Thousands of GPCR research studies have utilized heterologous expression systems such as human embryonic kidney cells (HEK293). Though often treated as 'blank slates', these cell lines nevertheless endogenously express GPCRs and related signaling proteins. The outcome of a given GPCR study can be profoundly influenced by this largely unknown complement of receptors and/or signaling proteins. Little easily accessible information exists that describes the expression profiles of the GPCRs in cell lines. What is accessible is often limited in scope - of the hundreds of GPCRs and related proteins, one is unlikely to find information on expression of more than a dozen proteins in a given cell line. Microarray technology has allowed rapid analysis of mRNA levels of thousands of candidate genes, but though often publicly available, the results can be difficult to efficiently access or even to interpret. RESULTS: To bridge this gap, we have used microarrays to measure the mRNA levels of a comprehensive profile of non-chemosensory GPCRs and over a hundred GPCR signaling related gene products in four cell lines frequently used for GPCR research: HEK293, AtT20, BV2, and N18. CONCLUSIONS: This study provides researchers an easily accessible mRNA profile of the endogenous signaling repertoire that these four cell lines possess. This will assist in choosing the most appropriate cell line for studying GPCRs and related signaling proteins. It also provides a better understanding of the potential interactions between GPCRs and those signaling proteins.

Laserspray ionization, a new method for protein analysis directly from tissue at atmospheric pressure with ultrahigh mass resolution and electron transfer dissociation.

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 µm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.

Dimerization of G protein-coupled receptors: CB1 cannabinoid receptors as an example.

A polyclonal antibody directed towards the last 73 amino acid residues of the rat type 1 cannabinoid (CB1) receptor strongly and exclusively labels a high molecular weight (between 160 and 200 kDa) form of the receptor in Western analysis. In contrast, a human CB1 polyclonal antibody identifies both monomeric CB1 as well as the high molecular weight form. The carboxy terminus (CT) antibody was also used in immunocytochemistry of rat hippocampal sections. Sections probed with CT antibody show intense staining of a meshwork of fibers and occasional interneurons of the stratum oriens, stratum pyramidal, and stratum radiatum of the CA1 and CA3 regions while mossy fibers and granule cells of the internal stratum appear unstained. These data provide evidence that CB1 likely exists as a dimer in vivo and that the carboxy end of the receptor may play a role in the assembly of the oligomer.

Cloning and molecular characterization of the rat CB2 cannabinoid receptor.

The rat peripheral cannabinoid receptor (rCB2) was cloned from a Sprague-Dawley rat spleen cDNA library and when translated, encodes a protein of 410 amino acids. Alignment of rCB2 with mouse (mCB2) and human (hCB2) peripheral cannabinoid receptors reveals a high degree of homology except in the carboxy terminus where rCB2 is 50 and 63 residues longer than hCB2 and mCB2, respectively. PCR screening and sequencing of rat genomic DNA showed that rCB2 is encoded by three exons interrupted by two introns, one of which is polymorphic and contains a 209 base pair B2 (SINE) element. By Northern hybridization and ribonuclease protection assay (RPA), rCB2 mRNA was detected in rat spleen, testis, thymus and lung but not in rat brain, heart, kidney or liver. Like hCB2 and mCB2 receptors, rCB2 activates mitogen-activated protein kinase when it is stably expressed in Chinese Hamster Ovary (CHO) cells. The importance of the carboxy terminus in regulating CB2 receptor desensitization and internalization is well-established. Thus, the profound differences identified in this region of the CB2 receptor between species mandates caution when extrapolating experimental results from non-human models to the effects of chronic CB2 receptor stimulation in humans.