CB₁ Cannabinoid Receptors and their Associated Proteins

Abstract

CB₁ cannabinoid receptor physiology, pathology and pharmacology

CB₁ receptors are G-protein coupled receptors (GPCRs) initially described as having such great abundance in brain tissue and neuronal cells, that the much lower levels in other tissues appeared to be of lesser significance [1]. In the early years of cannabinoid receptor characterization, pharmacological investigation of antinociception was emphasized because of the potential for a non-opioid analgesic, and pharmaceutical industry drug development programs were based upon this therapeutic opportunity [2,3]. This clinical application was thwarted as a result of the untoward side effects of memory impairment, cognitive dysfunction, and sedation [4,5], but has recently been reconsidered [6]. Although pharmaceutical development included treatment of nausea in cancer chemotherapy [7], the only CB₁agonists to succeed to market have been dronabinol (also known as •⁹-tetrahydrocannabinol (THC)) and nabilone [8–10].

Current research has made significant progress in clarifying the role of CB₁ receptors in such important aspects of cognition as reversal learning by which memory traces can be attenuated in the process of developing new patterns in response to novel relevant stimuli [11]. Highly coupled to memory and learning, CB₁receptors play a role in addiction processes that extend from reinforcement of high-fat, sweet food intake [12] to dependence on reinforcing drugs and alcohol [13–15]. CB₁ receptors abundant in the basal ganglia are implicated in motor dysfunction including Huntington’s disease and amyotrophic lateral sclerosis [16]. Other central nervous system diseases that might involve a CB₁ receptor component include schizophrenia, and bipolar, depression, and anxiety disorders [17,18]. To understand the mechanisms underlying these behavioral responses, we now know that CB₁ receptors are not only involved in neuronal synaptic remodeling as learning takes place, but also in neurogenesis, neuronal migration and appropriate axonal targeting and synaptogenesis as the brain develops [19–21].

The existence of CB₁ receptors in liver and adipose tissues was not appreciated until clinical trials of the CB₁ antagonist rimonabant (also known as SR141716A) for the treatment of obesity and dyslipidemias [22,23] uncovered the anomaly that decreases in adiposity in humans could not be entirely explained by a central nervous system effect of the drug to curtail food intake [24,25]. Current studies are focusing on both CB₁ and CB₂ receptors in hepatic metabolism and pathologies [26,27]. The interest in metabolic syndrome led to the expansion of studies of CB₁receptors on vascular as well as cardiac tissue [28] [29,30]. CB₁receptors are also relevant to many other organ systems including reproductive tissues [31], bone [32], and skin [33].

To summarize the relevant pharmacology, synthetic analogs of THC include classical cannabinoid agonists such as the highly potent and efficacious HU210, and the “non-classical” highly potent and efficacious agonists have a modified ring structure, such as CP55940 and CP55244 [34,35]. The aminoalkylindole CB₁agonists include the full agonist WIN55212-2. The endogenous ligands for the CB₁ receptor are a family of eicosanoid derivatives referred to as endocannabinoids, most notably 2-arachidonoylglycerol (2-AG) which behaves as a full agonist, and anandamide (arachidonylethanolamide), which behaves as a partial agonist. These CB₁ agonists have been reviewed previously [34–37], as well as in the present series (Mechoulam, 2010; Pertwee, 2010). CB₁ antagonists include the high-affinity aryl pyrazole, rimonabant, formerly known as SR141716, as well as other CB₁-selective antagonists of similar structure including AM251, taranabant, VCHSR and AM4113 [35,38–42]. LY320135 is a CB₁ antagonist having a structure that resembles the aminoalkylindole class of antagonists [43].

CB₁ receptor cellular signaling via G-proteins

Domains of the CB₁ receptor that selectively interact with Gi/o proteins have been identified [44]and appear to be coupled to Gαi/o proteins in the absence of exogenous agonists [45–47]. The juxtamembrane domain, which extends from transmembrane (TM) 7 to the cys palmitoylation site, forms a helix referred to as H8 [48–51]. This domain is able to trigger G-protein activation [44,48,52] via Gαo or Gαi3 [46,47,53]. CB₁ receptor mutants truncated at the membrane surface of the TM7 were not able to inhibit Ca²⁺ channels in response to CB₁ agonists [54]. The H8 amphipathic helix possesses cationic charges facing the G-protein surface that are important for agonist efficacy [48,50], with hydrophobic residues of the helix being critical for proper structural folding and positioning within the membrane bilayer [55]. A conformational change in the TM7 domain could displace the TM7-H8 elbow (see [51]. To test this, point mutations that replaced Leu at the elbow region with Phe or Ile were compared with the wild-type CB₁ receptor [56]. Human Embryonic Kidney 293 (HEK293) cells heterologously expressing the mutant receptors exhibited reduced agonist-stimulated [³⁵S]GTPγS binding to G-proteins, and the CB₁ receptor mutants failed to interact with Gαi3 although they could still bind Gαi1 and Gαi2 [56]. These CB₁ receptor mutants exhibited alterations in internalization in response to agonists [56]. The mutant receptors also exhibited significantly reduced inhibition of the Ca²⁺channels but a more rapid time course in response to WIN55212-2, which could be overcome by providing an abundance of the preferred G-protein, Gαo_A [56].

There is evidence to suggest that the CB₁ receptor IL3 facilitates association with Gαi1 or Gαi2 [44,46,47,53]. Helical structures at the N-terminal side of IL3 possessing a BBXXB-like motif characterize the potential for this domain to interact with G-proteins [57]. The IL3 helical region near H6 is believed to interact with Gαi1 [53]. A double mutation of Leu-Ala in the IL3-H6 junction switched the helical domain to a single turn structure, thereby converting the CB₁ receptor from interacting with Gi to interacting with Gs [53,58].

Signal transduction pathways utilized by CB₁ receptors have often been associated with interaction with Gi/o class of G-proteins as demonstrated by their sensitivity to pertussis toxin [37,59]. Inhibition of adenylyl cyclase, activation of the mitogen activated protein kinase (MAPK) family of kinases, inhibition of voltage-gated Ca²⁺ channels, and activation of inwardly rectifying K⁺currents (K_ir) are all signaling pathways that are pertussis toxin-sensitive (see reviews [34,37]. Regulation of ion channels by CB₁receptors are the direct result of G•• release upon Gi/o activation [60–64]. N-type voltage-gated Ca²⁺ currents were inhibited in differentiated neuroblastoma cells [62,63,65–67] and P/Q-type Ca²⁺ currents were inhibited in rat cortical and cerebellar neurons and cultured AtT-20 pituitary cells heterologously expressing CB₁receptors [68,69]. L-type Ca²⁺ currents in cat brain arterial smooth muscle cells were inhibited by CB₁ agonists, although the mechanism may not directly involve G•• [70].

CB₁ receptor interaction with proteins other than G-proteins

The diverse intracellular localization of endogenously-expressed CB₁ receptors was reported in studies using antibodies that recognize an N-terminal epitope of the receptor [125]. Immunocytochemical localization and sub-cellular fractionation studies indicated that a predominance of endogenously expressed CB₁ receptor in neuroblastoma cells reside in internal organelles, particularly along the peri-nuclear membranes [125]. Constitutively synthesized CB₁ receptors that had been pulse-chase labeled with [³⁵S]-amino acids were degraded via at least two different mechanisms having elimination half-lives of approximately 5 hr (70% of receptors) and >24 hr (30% of receptors) in cells that had not been exposed to agonist ligands, suggesting the presence of multiple pools of functional receptors within neuronal cells [125]. Cellular sorting of CB₁ receptors heterologously expressed in CHO-CB₁ cells was recognized by FACS and confocal microscopy to transit from internal stores to the plasma membrane in response to the antagonist rimonabant and from plasma membrane to intracellular compartments in response to agonist CP55940 [126]. Pretreatment with CP 55940 resulted in rapid desensitization of the CB₁ receptor agonist-mediated inhibition of forskolin-stimulated adenylyl cyclase or MAPK activation. Pretreatment of CHO-CB₁ cells with rimonabant resulted in significant augmentation of CP55940-mediated activation of MAPK, but had no significant effect on CP55940-mediated inhibition of forskolin-stimulated adenylyl cyclase (Rinaldi-Carmona et al., 1998). Internalization of CB₁receptors from neuronal plasma membranes at the soma has been proposed to be a mechanism by which these receptors can be diverted to plasma membrane loci along neuronal extensions [127–129]. To add to the complexity of interpreting data, many studies have been performed using exogenously expressed CB₁receptors that are fusion proteins with a fluorescent protein at the C-terminal tail, or else localization was determined using antibodies against epitopes in the C-terminal tail. Methods of detection that depend upon recognition of the C-terminal differ in their results when compared with endogenously expressed receptors having an accessible C-terminal for interaction with accessory proteins (see diversity of responses reported in [130]). Thus, it appears that domains along the CB₁ receptor C-terminal have the potential to alter the dynamics of cellular trafficking and signal transduction (summarized in Table 1). CB₁ receptors located at internal organelles may be of particular importance in autocrine regulation of cellular functions, particularly as sites of anandamide synthesis and degradation may be localized to intracellular membranous organelles (enzymes reviewed by [131]). Both cellular localization and function may be directed by CB₁receptor association with a variety of interacting proteins.

Table 1

Role of CB₁ Receptor-Associated Proteins in Protein Trafficking and Signal Transduction

Interaction of the CB₁ receptor with Cannabinoid Receptor Interacting Protein 1a (CRIP1a)

The CB₁ receptor has recently been shown to interact with a novel protein, the cannabinoid receptor interacting-protein, CRIP1a/b, within its C-terminus domain. CRIP1a/b were discovered by the Lewis laboratory, which reported that deletion of the CB₁ receptor C-terminal tail slowed the time to peak Ca²⁺ current inhibition, augmented the tonic inhibition of Ca²⁺ currents, and promoted the ability of the CB₁ receptor to sequester G-proteins [54,149]. They hypothesized that the C-terminal tail could serve an auto-inhibitory function. In seeking an accessory protein to regulate this activity, they used the CB₁ receptor distal C-terminal as bait in a yeast two-hybrid screen to identify a pair of splice variant proteins, CRIP1a and CRIP1b [150]. CRIP1a could bind to a GST-CB₁-C-terminal tail fusion protein, and could be co-immunoprecipitated with the CB₁ receptor [150].

The gene for CRIP1a and CRIP1b, is located on human chromosome two and consists of 3 coding exons. Both CRIP1a and CRIP1b have highly conserved sequence homology due to the fact that both proteins are encoded by exons 1 and 2. However, alternative splicing of exon 3 leads to the sequence variation and naming observed between these two isoforms; exon 3 encodes for amino acids 111–128 in CRIP1b and 111–164 in CRIP1a. Currently, both CRIP isoforms have been cloned and sequenced for human, rat and mouse, with significant homology seen among the rodent species. However, an alternative splicing variation has been found in mouse cerebellar tissue, which like CRIP1a/b is encoded for by exons 1 and 2, but also includes the noncoding regions of the 5′ end of exon 1 and the 3 prime end of exon 2. Genomic database searching has identified CRIP1a in all vertebrates, but CRIP1b is only found in primates indicating recent evolutionary processing of this gene [150]. In addition, CRIP1a distribution in mouse brain reveals co-expression with CB₁ in excitatory glutamatergic neurons, but not in inhibitory GABAergic interneurons [151,152].

CB₁ receptor mutagenesis studies show that the last 9 amino acids on the C-terminus of CB₁ are the minimum residues required for CRIP1a/b binding [150]. Furthermore, both isoforms require amino acids 34–110 (comprising exons 1 and 2) to interact with the CB₁. Because neither of the CRIP proteins interacts with the CB₂ receptor, they possess a unique and specific means for modulating the physiological effects mediated through the CB₁receptor. Alternative splicing isoforms that bind to CB₂ have yet to be discovered, and if identified may serve to selectively and differentially alter CB₁ and CB₂ signaling.

To date, little is known about the functional relevance of the CRIP1 family of proteins due to the lack of a three-dimensional model and insufficient sequence homology to other known proteins. A major difference between CRIP1a and CRIP1b is that CRIP1a contains a palmitoylation site and a C-terminus PDZ class I ligand. Site specific palmitoylation is a known modification that many adaptor and scaffolding molecules undergo and serves to regulate the function and trafficking of G-proteins, GPCRs and Src family kinases [153]. Proteins that contain PDZ ligands are believed to have a role in determining the cellular distribution of proteins that possess PDZ domains that recognize them. The functional relevance of CRIP1a having a PDZ ligand is intriguing, as it may allow CRIP1a to 1) interact with other proteins and act as a scaffolding site to establish variations in signal transduction, 2) enable the formation of homo/heterodimerization between CB₁and/or other receptors, and 3) modulate CB₁ trafficking events such as localization, desensitization, or internalization.

Studies using superior cervical ganglion neurons (SCG) stably transfected with CB₁ receptors have shown that the •• subunits of the trimeric G-protein complex can directly interact with N-type Ca²⁺ channels to inhibit Ca²⁺ influx [60]. This effect can be reversed by the antagonist/inverse agonist rimonabant, indicating that CB₁ receptors release G•• subunits to suppress N-type Ca²⁺channel activity. CRIP1a, but not CRIP1b, attenuated the inhibitory effects of CB₁ on N-type Ca²⁺ channels [150]. However, WIN55212-2 stimulation of CB₁-mediated inhibition of N-type Ca²⁺ channels was unaltered by CRI P1a. These results taken together suggest that CRIP1a/b may functionally modulate CB₁signal transduction in an agonist-independent manner. Additional research to define the effects of CRIP1a/b on other cannabinoid agonist and antagonist-regulated signal transduction will further delineate these mechanisms.

Many GPCRs can initiate agonist-independent regulation of signal transduction pathways, in which case ancillary proteins that internally bind to GPCRs can be key modulators of receptor-mediated events. There is also great variation in the C-termini of GPCRs, allowing for diverse and differential interactions, post-translational modifications, and trafficking seen within this superfamily of transmembrane proteins. Additionally, protein-protein interactions positively modulate GPCR signaling by influencing ligand-binding affinity and specificity, coupling between receptors, G-proteins and effectors, or targeting to specific subcellular locations. Receptor-interacting-proteins like Homers and dopamine receptor interacting proteins (DRIPs) regulate the intracellular activity of GPCRs using various mechanisms. For example, metabotropic glutamate receptors (mGluRs) are known to interact via their C-terminus with a family of proteins called Homers. Homer proteins have been implicated in a variety of roles including trafficking of type I mGluRs and receptor-mediated signal localization [154]. This class of proteins is subdivided into three families (1, 2, 3), and similar to what is seen in CRIP1a/b, families 1 and 2 are splice variations of the same gene. For the most part, Homer proteins are constitutively expressed and have two functional sites: an amino-terminal Enabled/Vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domain and a coiled-coil leucine zipper carboxy-terminus. In addition to these two functional sites, Homers possess a PDZ domain that has been shown to bind with PSD-95, GKAP and Shank1 and 3 [155]. One important aspect on mGluR receptors is their ability to function as dimers, and therefore, the Homer family of proteins may participate in this process [156]. Homers are also known to form a complex between mGluRs and IP3 receptors [154]. Thus, if CRIPs exhibit functional homology to Homer proteins, then CRIPs may play a pivotal role in localization of the CB₁ receptor to its signaling partners, such as voltage-gated Ca²⁺ channels and G-protein linked K⁺channels. However, unlike CB₁ receptors, the majority of mGluRs are located at post synaptic density areas where Homers can be up-regulated by neuronal activity [154]. An example of this is that expression of Homer 1a is induced upon neuronal excitation [157], and due to the lack of a binding domain in its C-tail, can act as a dominate-negative protein by disrupting interactions between mGluR receptors and other proteins. In HEK293 cells, expression of Homer1a, but not its isoform Homer-1b, resulted in increased mGluR translocation to the plasma membrane. Studies suggest that Homer 1b influences mGluR 1 and 5 retention in intracellular stores until the up-regulation of Homer 1a evokes transit of receptors to the plasma membrane [158]. Because Homer 1a and CRIP1b both lack functional domains in their C-tail, they may perform similar functions as dominant negative regulators.

Like CB₁ receptors, D₂ dopamine receptors are GPCRs that are predominately expressed on presynaptic terminals, couple to Gi/o proteins, and initiate similar signal transduction pathways. D₂receptors associate with interacting-proteins (DRIPs) that bind to the C-terminus of the receptor. The D₂ receptor DRIP neuronal Ca²⁺ sensor 1 (NCS-1) inhibits D₂ receptor desensitization in a Ca²⁺-dependent fashion by binding to GRK2 and preventing its phosphorylation of the D₂ receptor [159]. Because CRIP1a/b bind to a domain near the CB₁ receptor internalization site, CRIP may serve a similar function asNCS-1. To date, other dopamine receptor interacting-proteins (calcyon and DRIP78) have also been identified. The endoplasmic reticulum membrane-bound protein DRIP78 regulates the transport of many GPCRs (D1, M2, AT1), via their C-tail, to the plasma membrane [160]. Both sequestration and overexpression of DRIP78 results in D₁ and •₂AR localization in the endoplasmic reticulum, along with reduced ligand binding and receptor glycosylation [161].

The further identification of CRIP proteins as well as their functional importance will provide a greater understanding into the regulation and neurotransmission of cannabinoid receptors. Because CRIP1a colocalizes with CB₁ signaling at excitatory synapses, it is a novel target for the treatment of disorders associated with excessive excitatory transmission, such as epilepsy [152]. Thus it will be important to continue research into the CRIP proteins to elucidate the mechanisms involved in CRIP1a/b modulation of CB₁ receptors and the opportunity that this offers in the development of cannabinoid based drugs.

Interaction of the CB₁ Receptor with Factor Associated with Neutral sphingomyelinase (FAN)

Signal transduction via regulation of the second messenger ceramide can be regulated by CB₁ receptors either via sphingomyelin hydrolysis or by de novo synthesis of ceramide (see review [162,163]). To summarize, transient increases in intracellular ceramide are regulated by the interaction of the CB₁receptor with neutral sphingomyelinase via the interacting protein FAN. Once released into the cell, this short-lived production of ceramide can regulate cellular metabolic processes. The enzymatic production of ceremide de novo is under the CB₁ receptor regulation of serine palmitoyltransferase or ceramide synthase, and initiates signaling pathways leading to cyclooxygenase-2 expression and apoptosis in cells that are susceptible to this mediator [164–166].

FAN directs the agonist-stimulated CB₁ receptor to the regulation of sphingomyelin hydrolysis, leading to the release of ceramide [167]. In an astrocyte model, CB₁ agonists promoted a complex of FAN with CB₁ receptors, and this could be blocked by the CB₁antagonist rimonabant, but not by pertussis toxin treatment, indicating that Gi/o proteins were not intrinsic to the process [167]. The requirement for FAN is based upon the attenuation of the ceramide generation in cells that express a dominant negative form of this protein[167]. CB₁ receptors and FAN could co-immunprecipitate, demonstrating their interaction as a complex. Studies based upon FAN interactions with other proteins suggest that the C-terminal of the CB₁ receptor is the site of a putative neutral sphingomyelinase activation domain [167]. Interestingly, FAN is a WD40 repeat protein, as is the G• subunit of G-proteins and many other adaptor proteins [163]).

Sphingomyelin hydrolysis in cultured astrocytes or glioma cells yielded an increase in intracellular ceramide within 15 min [87,168]. CB₁-mediated release of ceramide activates the Raf-1-MEK-ERK pathway to regulate glucose metabolism [86]. The Guzman laboratory has speculated that this ceramide-signaling pathway may serve an important function of astrocytes to supply metabolic substrates to neurons for oxidative metabolism at synapses and other sites having rapid biosynthetic requirements [163].

Perspectives

The majority of the data regarding cellular mechanisms for CB₁receptor actions have been based upon experiments performed in the brain or in neuronal models (Table 1). As we now are learning about CB₁ receptors in many other cell types, it is important to recognize that cellular signal transduction for highly specialized cells may deviate from what we have learned regarding neurons. Neurons are excitable cells, and are structurally unique in their cell-cell interactions (i.e., they are polarized in the structure of axons and dendrites, and form unique sites of interaction, the synapse). Other cell types possess their unique abilities to respond to hormones and other mediators to regulate their specific functions within a tissue or organ. While we can use our present knowledge as a guide, we certainly cannot expect that all cell types will respond to CB₁ receptor stimulation via activation of the same signaling pathways. This notion of diversity leads us to speculate that agonists and allosteric modulators might be discovered or developed which can trigger the regulation of unique, cell-specific responses.

Footnotes

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