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Correspondence: Address to Andrea G. Nackley, PhD, Center for Pain Research and Innovation, University of North Carolina, Room 5506, Koury Oral Health Sciences Bldg, 385 S Columbia St, Chapel Hill, NC 27599-7455.
Drugs that target G protein–coupled receptors (GPCRs) represent the primary treatment strategy for patients with acute and chronic pain; however, there is substantial individual variability in both the efficacy and adverse effects associated with these drugs. Variability in drug responses is due, in part, to individuals’ diversity in alternative splicing of pain-relevant GPCRs. G protein–coupled receptor alternative splice variants often exhibit distinct tissue distribution patterns, drug-binding properties, and signaling characteristics that may impact disease pathology as well as the extent and direction of analgesic effects. We review the importance of GPCRs and their known splice variants to the management of pain.
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Learning Objectives: On completion of this article, you should be able to (1) explain the importance of G protein–coupled receptors to pain signaling and modulation, (2) explain the basic concepts of alternative splicing, and (3) describe how individual variability in alternative splicing of G protein–coupled receptors may contribute to variability in the nature of pain as well as responses to analgesic drugs.
Disclosures: As a provider accredited by ACCME, Mayo Clinic College of Medicine (Mayo School of Continuous Professional Development) must ensure balance, independence, objectivity, and scientific rigor in its educational activities. Course Director(s), Planning Committee members, Faculty, and all others who are in a position to control the content of this educational activity are required to disclose all relevant financial relationships with any commercial interest related to the subject matter of the educational activity. Safeguards against commercial bias have been put in place. Faculty also will disclose any off-label and/or investigational use of pharmaceuticals or instruments discussed in their presentation. Disclosure of this information will be published in course materials so that those participants in the activity may formulate their own judgments regarding the presentation.
In their editorial and administrative roles, William L. Lanier, Jr, MD, Terry L. Jopke, Kimberly D. Sankey, and Nicki M. Smith, MPA, have control of the content of this program but have no relevant financial relationship(s) with industry.
Dr Maixner is a co-founder and equity stock holder in Algynomics, Inc, a company providing research services in personalized pain medication and diagnostics, and a patent holder with UNC Proove Bioscience. In addition, he receives consulting fees from National Institutes of Health, APS, and Orthogen with research funding support from National Institutes of Health/National Institute of Dental and Craniofacial Research.
Dr Nackley receives research support from the following sources: National Institutes of Health/National Institute of Neurological Disorders and Stroke R01 NS072205 (Principal Investigator); National Institutes of Health/National Institute of Neurological Disorders and Stroke P01 NS045685 (Principal Investigator); National Institutes of Health/National Institute of Dental and Craniofacial Research U01 DE017018 (Investigator); National Vulvodynia Association (NVA) (Principal Investigator); and UNC NCTraCS 50KR81417 (Principal Investigator).
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Date of Release: 8/01/2015
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Nociceptive pain is an acute response to environmental stimuli that warns of potential or actual tissue damage. In the event of actual damage, inflammatory and/or neuropathic pain may occur. Inflammatory pain occurs in response to damage of tissues and infiltration of immune cells, while neuropathic pain occurs in response to damage of nerves. Inflammatory and neuropathic pain typically serve to promote wound healing and repair; however, in many cases, the pain outlasts the stimulus and becomes chronic. Unlike inflammatory and neuropathic pain, functional or idiopathic pain is characterized by perpetual abnormalities in sensory processing that occur in the absence of direct inflammation or nerve damage.
Acute and chronic pain are primarily treated with pharmacological agents that promote analgesia. The principle target of a variety of analgesic drugs including opioids, cannabinergics, and antidepressants is G protein–coupled receptors (GPCRs). On activation, GPCRs initiate molecular changes resulting in excitation or inhibition of nerve, immune, and glial cells important for the onset and maintenance of pain. Although the critical role of GPCRs in pain biology and management is well established, reliably effective therapeutics with minimal adverse effects are lacking. Interindividual variability in response to a given analgesic is largely due to variation at the genetic level. Of particular interest are genetic variants in alternative splice regions that alter protein coding of the messenger RNA (mRNA), giving rise to proteins that differ in form and function (ie, alternative splice variants). This review highlights the importance of alternative splicing in the regulation of GPCRs involved in the transmission and modulation of pain.
GPCRs Are Relevant for the Treatment of Pain
The human genome encodes approximately 800 distinct GPCRs, 70% of which contribute to pain or pain-related phenotypes.
G protein–coupled receptors interact with a tremendous variety of signaling mediators, ranging from small molecules to large peptides and proteins. Although each receptor has the ability to induce a range of functional intracellular changes, all GPCRs possess a distinct and evolutionarily conserved architecture. Each canonical or classic receptor comprises 7 transmembrane (TM) proteins that span the cellular membrane. These TM proteins are interconnected by intracellular and extracellular loops (Figure 1). In addition, there are amino acid chains known as N-terminus and C-terminus tails, which are attached to the first and last TM, respectively. As alluded by its name, every GPCR is coupled to a G protein, which acts as a molecular switch to regulate cellular activity (Table 1).
Figure 1G protein–coupled receptor (GPCR) structure and function. A, G protein–coupled receptors are composed of 7-transmembrane domains (gray) interconnected by 3 intracellular (orange) and 3 extracellular (purple) loops. On the end of the first and last transmembranes are the N-terminus (blue) and C-terminus (red), respectively. As its name suggests, a GPCR is bound to a trimeric G protein composed of α and β/γ subunits. B, When a ligand (black circle) binds to a GPCR, the associated G protein separates into the α and β/γ subunits. These subunits then stimulate a variety of downstream effectors that produce changes in cellular activity (see Table 1).
The resulting structure created by the TM segments and loops provides interactive sites where ligands can bind. Ligands that bind to their receptor and initiate cell signaling are referred to as agonists. On binding, agonists produce a conformational change of the GPCR and subsequent uncoupling of the associated G protein. Once uncoupled, the G protein separates into 2 subunits (the α and β/γ subunits), each of which initiates a chain of molecular reactions that affect cellular activity.
Depending on the type of G protein, the initiated downstream effects can promote cellular excitation or inhibition (Table 1). In general, agonists that activate pain-relevant GPCRs coupled to Gs typically produce pain, while those coupled to Gi typically inhibit pain.
Other ligands, known as antagonists, compete with agonists for the GPCR binding site and impede G protein uncoupling and downstream signaling events. Because of their ability to modulate cellular activity at each step of the pain pathway, GPCRs represent a popular pharmacological target for the management of clinical pain. In fact, over 60% of commonly prescribed analgesics work by binding to GPCRs.
Table 2 provides a summary of these GPCRs (opioid, cannabinoid [CB], adrenergic, and serotonergic receptors) along with their associated G protein, endogenous ligands, and analgesic compounds.
Table 2GPCRs Commonly Targeted for Clinical Pain Management
Opioid receptors are among the most well-known GPCRs that regulate the transmission and perception of pain. There are 4 opioid receptor subtypes: the μ-opioid receptor (MOR-1), the δ-opioid receptor, the κ-opioid receptor, and the nociceptin receptor. Of these subtypes, MOR-1 is the classic receptor responsible for analgesic responses to endogenous endorphins as well as exogenous drugs. On agonist binding to MOR-1, its associated Gαi protein is activated and produces cellular inhibition of pronociceptive neurons.
For this reason, opioids are used in the management of acute pain (such as that associated with surgery) as well as chronic pain disorders such as low back pain, extremity pain, and osteoarthritis.
Opioid antagonists, usually coadministered with opioid agonists to reduce the development of unwanted opioid effects, are also capable of producing analgesia independently of MOR-1.
Cannabinoid receptors share similar signaling properties with MOR-1, making them attractive targets for clinical pain management. There are 2 CB receptor subtypes, CB1 and CB2, both of which couple to Gαi. Cannabinoid receptors play an important role in promoting analgesia in response to endocannabinoids such as 2-arachidonoylglycerol and anandamide. Commercially available CB agonists such as nabilone and tetrahydrocannabinol, which bind to both CB subtypes, are used to treat fibromyalgia and neuropathic pain.
Adrenergic receptors (ARs), which mediate the physiologic responses to epinephrine and norepinephrine, represent another frequently targeted class of GPCRs. The adrenergic superfamily includes 3 subtypes respectively of α1-ARs (α1A-AR, α1B-AR, α1D-AR), α2-ARs (α2A-AR, α2B-AR, α2C-AR), and β-ARs (β1-ARs, β2-ARs, β3-ARs). The α2-AR couples to Gαi and promotes analgesia via cellular inhibition. Hence, α2-AR agonists such as trazodone are used to promote analgesia. In contrast, α1-AR, which is coupled to Gαq, facilitates cellular excitation of pronociceptive neurons, resulting in increased pain signaling. The β-ARs also facilitate pain signaling via Gαs signaling. To attenuate their excitatory contributions, α1-AR and β-ARs are commonly used to treat a range of chronic pain disorders such as migraine, neuropathic pain, and fibromyalgia.
Finally, serotonin receptors, which mediate physiologic responses to the monoamine serotonin (or 5-hydroxytryptamine [5-HT]) play an important role in pain management.
The serotonin superfamily is quite large, including 7 general members: 5-HT1 (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F), 5-HT2 (5-HT2A, 5-HT2B, 5-HT2C), 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7. With the exception of the 5-HT3 receptor, a ligand-gated ion channel, all 5-HT receptors are GPCRs. The effects of the 5-HT receptor family on pain are heavily dependent on the receptor subtype. Triptans target Gαi-coupled 5-HT1 receptors, which promote analgesia via cellular inhibition, and normalize vascular changes associated with migraine.
Antidepressants promote chronic synaptic serotonin release that causes the down-regulation of Gαq-coupled 5-HT2 receptors, thus attenuating their excitatory contributions to pain signaling. The 5-HT antagonists that target 5-HT4 receptors in the central nervous system and the gastrointestinal (GI) tract are used in the treatment of migraine
Meanwhile, the net effect of 5-HT7 activation on pain is highly dependent on the location of the receptor. Activation of 5-HT7 receptors on peripheral nerve terminals produces pain,
Additionally, their use is constrained by adverse effects, such as altered mental state, nausea, constipation, sedation, and life-threatening respiratory depression. Variability in patient response and adverse effect profiles is due, in part, to diversity in alternative splicing of GPCRs expressed in tissues that regulate pain processing. By expanding our understanding of GPCR alternative splice variants and their associated pharmacodynamic responses, we will be able to better predict patient-centered treatment outcomes.
Alternative Splicing Adds to the Diversity of GPCR Signaling
Alternative splicing is an important mechanism of gene regulation, affecting approximately 90% of all genes within the human genome.
A single gene is able to generate exponential protein coding capabilities via alternative splicing. Before alternative splicing, a gene is first transcribed into precursor mRNA (pre-mRNA). The pre-mRNA sequence contains short-protein coding regions known as exons. Interspersed between the exons are longer noncoding regions known as introns (Figure 2). Before the sequence can be translated to produce protein, the introns and alternative exons within pre-mRNA are removed, or spliced, and the constitutive exons are brought together, resulting in the canonical mRNA transcript ready for protein synthesis. When alternative splicing occurs, however, the pre-mRNA is edited such that constitutive exons are removed from, or introns are retained in, the final mRNA transcript. The most common type of alternative splicing within the human genome is exon skipping,
in which constitutive exons are excluded from the final mRNA transcript. Another common type of alternative splicing is splice site selection, in which the portion of an exon is spliced out because of the presence of a nucleotide sequence that facilitates splicing activity.
Intron retention is another type of alternative splicing in which an intron remains in the final mRNA transcript. Each type of alternative splicing will render an mRNA transcript and corresponding protein that is structurally different from the canonical protein produced from the standard template (Figure 3).
Figure 2Different types of alternative splicing. The most common type of alternative splicing in animals is exon skipping (A), in which a constitutive exon is spliced from the final messenger RNA (mRNA) transcript. Alternative 3′ (B) and 5′ (C) splice sites provide additional junctions within an exon, resulting in partial splicing of the exonic mRNA sequence. D, Intron retention is a rare type of alternative splicing that occurs when an intron remains within the final mRNA transcript.
Figure 3Structural variations in G protein–coupled receptors (GPCRs) as a result of alternative splicing. Exons within the messenger RNA transcript serve as coding regions for specific sections of protein. Alternative splicing events that change or remove exonic sequences can produce GPCR splice variants with corresponding changes in protein composition and/or structure. A, Splicing events that lead to alterations in exon 1 can yield GPCRs with truncated N-termini that affect ligand binding, while events that lead to alterations in exon 4 can yield GPCRs with truncated C-termini that affect G protein coupling and signaling. B, Splicing events can also lead to skipping of an exon that codes for a unit of the GPCR, such as a transmembrane, thus yielding a truncated GPCR lacking the encoded section, such as a 6-transmembrane (6-TM) splice variant.
Accumulating evidence suggests that alternative splicing substantially adds to the functional diversity of the human genome and that variations in these processes produce pathologic states.
Allelic variants that alter the ratio of functionally distinct protein isoforms through alternative splicing may produce changes in the direction of pain-relevant GPCR pharmacodynamics (eg, coupling to stimulatory vs inhibitory G protein effector systems), yet remain understudied. A PubMed search of the term alternative splicing pain yields only 87 relevant original research articles. Most are focused on ion channels such as voltage-gated calcium channels
with only 12 articles focusing on GPCRs. This is an important area of study because identification of GPCR splice variants differentially expressed in individuals with altered pain perception and/or analgesic responses will help elucidate novel targets for the development of individualized treatment strategies.
Functional GPCR Alternative Splice Variants
Examples of alternative splice variants of pain-relevant GPCRs that exhibit diversity in expression and signaling profiles include the aforementioned MOR-1, CB receptors, adrenergic receptors, and serotonin receptors. Of additional interest are nociceptin, prostaglandin, and neurokinin receptors, which are not targeted by common analgesics but are critical for the induction and modulation of pain. Accumulating evidence from in vitro, preclinical, and clinical studies suggests that alternative splicing of these and other GPCR transcripts adds additional layers of complexity to GPCR signaling and pharmacodynamic responses (Table 3).
Table 3Signaling, Tissue Distribution, and Function of Known GPCR Splice Variants
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in Alzheimer's disease brains.
COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord.
Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands.
Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands.
Acute agonist-mediated desensitization of the human α1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of α1aAR splice variants.
Acute agonist-mediated desensitization of the human α1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of α1aAR splice variants.
Acute agonist-mediated desensitization of the human α1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of α1aAR splice variants.
Acute agonist-mediated desensitization of the human α1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of α1aAR splice variants.
Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon: RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.
The pharmacological manipulation of MOR-1 is an essential component of clinical pain treatment. Although the signaling characteristics of MOR-1 are well established, we are just beginning to understand the complex nature of genetic variants that contribute to alternative splicing. At least 20 MOR-1 splice variants have been identified in mouse and human genomes,
provide evidence that the gene expression of MOR-1 splice variants represent compensatory responses to long-term opioid administration that stabilize or diminish the development of tolerance. Other studies have found that the presentation of some unwanted effects are due to the activation of MOR-1 splice variants. For example, Liu et al
have documented that because of its distinct C-terminus, the splice variant MOR-1D dimerizes with the gastrin-releasing peptide receptor in the mouse spinal cord to produce opioid-induced itch. Another splice variant known as MOR-1K, a truncated receptor lacking the N-terminus and first TM, has been implicated in the paradoxical increase in pain sensitivity known as opioid-induced hyperalgesia (OIH). In contrast to MOR-1, which typically couples to Gαi, MOR-1K couples to Gαs to activate adenylate cyclase (AC) and increase intracellular calcium, thus engaging pronociceptive signaling events that likely drive OIH.
Oladosu F, O'Buckley S, Nackley AG. Elucidating the role of MOR-1K in opioid-induced hyperalgesia via siRNA gene knockdown. International Association for the Study of Pain Conference, October 6-11, 2014, Buenos Aires, Argentina. International Association for the Study of Pain. 2014.
Additional studies investigating the functional characteristics of MOR-1 splice variants provide evidence that a set of these receptors promote opioid analgesia by providing exclusive binding sites for different opioids. Transgenic mice lacking exon 11, an exon that provides an alternative promoter region for the MOR transcript, have substantial reductions in the analgesic efficacies of heroin, fentanyl, and the morphine metabolite morphine-6β-glucuronide,
suggesting that exon 11–containing variants play a critical role in opioid analgesia. Exon 11–containing splice variants also mediate the analgesic effects of iodobenzoylnaltrexamide (IBNtxA), a novel synthetic opioid that produces 10 times the analgesic efficacy of morphine without producing respiratory distress, dependence, tolerance, or GI distress in rodents.
MOR-1 splice variants also promote analgesia by enhancing canonical receptor function. Single-TM splice variants MOR-1R and MOR-1S structurally enhance MOR-1 function by stabilizing the canonical 7-TM (the GPCR as a whole, indicating the total number of transmembrane segments the receptor has) receptor at the cellular membrane.
Collectively, these studies highlight the importance of MOR-1 alternative splice variants in mediating opioid analgesia, as well as adverse effects such as tolerance, itch, and OIH.
Although few preclinical studies have examined the opioid receptor-like nociceptin receptor (ORL-1), it may also play an influential role in opioid analgesia. Majumdar et al
found that the exon 11 splice variant MOR-1G dimerizes with ORL-1 to provide a binding site for novel opioid IBNtxA, suggesting that ORL-1 interacts with MOR-1 splice variants to provide specific opioid binding sites. The contribution of ORL-1 to splice variant signaling is further complicated by the existence of its own splice variants, ORL-1Long and ORL-1Short.
Thus far, ORL-1Short has been implicated in the regulation of the canonical receptor, indicating a possible influence over ORL-1 function.
CB Receptors
Both the CB1 and CB2 receptors undergo alternative splicing to yield variants differing at their N-terminal region. The CB1A variant is truncated by 61 amino acids, with the first 28 amino acids completely different from the canonical CB1.
Although its tissue distribution largely overlaps with that of CB1, CB1A exhibits decreased agonist binding and activity, which might be due to a lack of 2 glycosylation sites typically important for signal transduction.
The CB1B variant lacks the first 33 N-terminus amino acids, and although it overlaps with CB1 in a number of tissues, its abundant expression in fetal brain suggests it may play an important role in development.
Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands.
The gene CB2A is initiated from the more distal promoter and includes exons 1a and 1b spliced to exon 3, while CB2B is initiated from the more proximal promoter and includes exon 2 spliced to exon 3. The CB2A variant is predominantly expressed in testes and at lower levels in spleen and brain. In contrast, the CB2B variant is predominantly expressed in spleen with very low expression in brain and no expression in testes. These tissue-specific distribution patterns may indicate specialized roles for the different splice variants with respect to pain modulation, immune response, and spermatogenesis.
Adrenergic Receptors
Adrenergic receptors play a key role in pain processing as well as cognition and cardiovascular function. While α2-ARs, β1-ARs, and β2-ARs are highly relevant to the modulation of pain by endogenous and exogenous agonists, the genes encoding these receptors are intronless and not subject to alternative splicing. Among the remaining adrenergic receptors, the α1a-AR subtype has been studied the most extensively with respect to alternative splicing.
The human ADRA1A gene locus comprises more than 8 exons and codes for 15 known splice variants.
The canonical receptor is generated through splicing exon 1 (coding for the N-terminus and TMs 1 to 6) together with exon 2 (coding for TM7 [the specific transmembrane of a GPCR that is coded by a specific exon] and the C-terminus). Four C-terminus splice variants (α1a-2, α1a-3, α1a-4, α1a-5) have been identified that are generated through the use of additional acceptor sites at varying locations within, and distal to, exon 2. The α1a-2, α1a-3, and α1a-4 variants exhibit ligand binding properties and tissue distribution profiles similar to α1a-AR, although α1a-3 and α1a-4 are absent in the kidney.
Acute agonist-mediated desensitization of the human α1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of α1aAR splice variants.
This diversity in α1a-AR signaling may contribute to differential responses to α1-AR antagonists used in the treatment of pain.
In addition, eleven 6-TM variants (α1a-6 through α1a-16) have been identified that are generated through exon skipping. These variants lack TM7, and their C-terminal tails are located extracellularly.
The truncated 6-TM variants are expressed in similar tissues as α1a-AR but are localized exclusively within the cell and unable to bind α1-AR agonists or directly mediate signal transduction. The 6-TM variants do, however, impair α1a-AR ligand binding and trafficking to the cell surface. Thus, α1a-AR 6-TM variants likely play an important physiologic role by modifying the function and expression of their parent 7-TM receptors.
One α1b-AR splice variant has also been identified in human brain.
The α1b-AR protein is generated through splicing of exons 1 and 2. In contrast to the canonical receptor, the α1a-2AR includes an immediately adjacent sequence following exon 1 in its coding sequence and excludes exon 2 that codes for TM7. Tseng-Crank et al
also identified low levels of a truncated ADRA1D transcript, but the result was inconclusive and naturally occurring α1d-AR variants were not observed. More work is required to determine the potential functional role of α1b-AR and α1d-AR variants.
The β3-AR is primarily known for its ability to regulate energy metabolism and thermogenesis,
The gene encoding β3-AR undergoes alternative splicing within the coding region to yield 2 C-terminal splice variants differing with respect to tissue expression, G protein signaling profiles, and regulatory properties.
The β3A-AR and β3B-AR splice variants contain completely unique terminal chains that are 13 and 17 amino acids long, respectively. The β3a-AR is primarily enriched in fat tissue and couples exclusively to Gαs, while the β3b-AR is primarily enriched in brain and couples to both Gαs and Gαi. In addition, the β3a-AR exhibits increased agonist-induced extracellular acidification, a measure of cyclic adenosine monophosphate–independent cellular activity. Their unique tissue distribution and signaling profiles, together with the known functional role of β3-ARs, could indicate that β3a-ARs play a greater role in lipolysis/thermogenesis and that β3b-ARs in brain mediate pain. Although these studies were conducted in mice, it is important to note that the human β3-AR contains a substantial number of genetic variants that are predicted to regulate alternative splicing.
Of the 5-HT1 (A, B, D-F), 5-HT2 (A-C), 5-HT4, 5-HT5, 5-HT6, and 5-HT7 GPCR family members, the 5-HT2A, 5-HT2C, 5-HT4, 5-HT6, and 5-HT7 receptors are known to undergo alternative splicing.
The human 5-HT2 receptor subtypes (5-HT2A, 5-HT2B, and 5-HT2C) couple to Gαq proteins to promote the transient release of intracellular calcium. One truncated splice variant of 5-HT2A (5-HT2A-tr) has been identified that utilizes alternate splice donor and acceptor sites to yield a 3-TM receptor with 57 unique amino acids in the C-terminal region.
The 5-HT2A-tr is coexpressed with 5-HT2A in most brain tissues but is unable to couple to the calcium pathway. Two truncated splice variants of 5-HT2C (5-HT2CT and 5-HT2C-R-COOHΔ) have also been identified. Similar to 5-HT2A-tr, the 5-HT2CT variant utilizes alternate splice donor and acceptor sites to yield a 3-TM receptor with 19 unique amino acids in the C-terminal region.
Compared with the canonical 5-HT2C receptor, the truncated variants exhibit similar expression patterns but have impaired 5-HT ligand binding and G protein coupling.
Agonists targeting 5-HT4 are beneficial in alleviating abdominal pain associated with IBS. Of all the 5-HT receptors, 5-HT4 possesses the greatest diversity in alternative splicing. At least 10 splice variants have been identified that vary with respect to their tissue distribution and function. Nine C-terminus variants (5-HT4a, 5-HT4b, 5-HT4c, 5-HT4d, 5-HT4e, 5-HT4f, 5-HT4g, 5-HT4i, 5-HT4n) have been identified that are identical up to amino acid Leu358, after which they vary in sequence and length.
Although all of the 5-HT4 splice variants display typical ligand binding properties, some have notable functional differences. Both of the GI-specific 5-HT4d and 5-HT4h variants have a tendency to recognize 5-HT antagonists as partial agonists.
Furthermore, the 5-HT4d variant exhibits a remarkable 20-fold increase in cyclic adenosine monophosphate formation following application of the 5-HT4 agonist renzapride.
The 5-HT4b variant is unique in its ability to couple to Gαi as well as Gαs proteins, suggesting its diverse signaling capabilities in the GI tract, brain, and other tissues.
The ability of GPCRs to increase basal AC activity has been reported previously and can result in physiologic functions of the receptor that are largely independent of endogenous ligands or exogenous drugs.
Collectively, these studies illustrate the high degree of tissue and signaling specificity for a number of 5-HT4 splice variants that may be attractive targets for the development of new, more selective drugs for the treatment of IBS among other conditions.
The 5-HT6 receptor is unique in that it is expressed almost exclusively in the central nervous system.
The corresponding receptor includes the TM1-3 and 10 unique amino acids in its C-terminus. In contrast to 5-HT6, the expression of 5-HT6-tr is limited to substantia nigra and caudate. The 5-HT6-tr receptor is able to translocate to the membrane, yet is unable to bind serotonin. This splice variant may have a yet-to-be-determined function or be indicative of abnormalities due to pathologic state.
The 5-HT7 receptor is expressed on primary afferent nociceptors, as well as in pain-relevant brain regions where it couples to Gαs to mediate the transmission and modulation of pain. Three splice variants of 5-HT7 (5-HT7a, 5-HT7b, 5-HT7d) have been identified that are all generated through alternative splicing of the second intron located near the C-terminal coding region. The 5-HT7a and 5-HT7b variants have tissue expression profiles and functional characteristics similar to the canonical receptor, although 5-HT7b has been found to exhibit considerably higher constitutive AC activity when expressed in stable cell lines.
and displays unique functional characteristics. Compared with the canonical 5-HT7 receptor and the 5-HT7a and 5-HT7b variants, the 5-HT7d variant displays agonist-independent internalization (even in the presence of antagonist) and associated reductions in agonist-induced AC activity.
It has been suggested that differences in the functional characteristics of 5-HT7 variants are due to specific features of their carboxyl tails, leading to differential interactions with protein partners that mediate their activity, trafficking, and/or internalization.
Prostaglandins, such as prostaglandin E2, are a product of cyclooxygenase that facilitates pain transmission through binding to the prostaglandin E receptor 3 (EP3 receptor). Activation of the Gαi-coupled EP3 receptor has been reported to produce analgesia
Functional evidence for interaction between prostaglandin EP3 and κ-opioid receptor pathways in tactile pain induced by human immunodeficiency virus type-1 (HIV-1) glycoprotein gp120.
These contradictory effects may be due to the presence of EP3 splice variants. To date, 6 C-terminus splice variants (EP3A through EP3F) have been identified. Of these, the EP3C receptor exhibits the most unique signaling characteristics because it is able to couple to Gαs as well as Gαi.
The dual coupling of the EP3C variant to different G proteins may explain the ability of EP3 ligands to produce both analgesia and hyperalgesia.
Neurokinin-1 Receptor
Neurokin-1 receptors (NK-1Rs) are targets for the endogenous propain ligand substance P. Their activation results in Gαq-mediated increases in intracellular calcium levels and production of proinflammatory cytokines.
Alternative splicing of the NK-1R yields a truncated variant (NK-1Rtruncated) that lacks the C-terminus and has functional properties that differ from the canonical receptor. Unlike NK-1R, activation of the NK-1Rtruncated variant does not result in increased levels of calcium or nuclear activity of factor κB. Instead, activation of NK-1Rtruncated results in decreased phosphorylation of protein kinase C and levels of interleukin 8. A recent clinical study reported the utility of an NK-1R antagonist in the treatment of chronic pain conditions and anxiety.
Neurokinin-1-receptor antagonism decreases anxiety and emotional arousal circuit response to noxious visceral distension in women with irritable bowel syndrome: a pilot study.
Results from functional studies of the NK-1Rtruncated variant suggest that splice variant–specific agonists may also be useful for pain management.
Clinical Relevance of Functional Gene Regulatory Events
Given the extensive list of alternative GPCR splice variants and their known impact on signaling and pharmacodynamics, it is expected that these variants have important clinical implications for pain management. Major strides in both preclinical and clinical research are still needed before we can reliably predict a patient’s treatment response on the basis of their splice variant expression profile. Such strides have been made, however, in the study of another type of gene variation, single-nucleotide polymorphisms (SNPs). Like alternative splicing, SNPs within key pain-related genes can result in changes that subsequently affect the encoded protein. For example, SNPs in the gene encoding catechol-O-methyltransferase (COMT) (an enzyme that metabolizes catecholamines) are indicative of abnormalities in COMT function and predictive of chronic pain risk and treatment response. Human genetic association studies have revealed that the rs4680 SNP, alone or in combination with other nearby SNPs, is predictive of temporomandibular disorder and fibromyalgia onset.
Tan E-C, Lim ECP, Ocampo CE, Allen JC, Sng B-L, Sia AT. Common variants of catechol-O-methyltransferase influence patient-controlled analgesia usage and postoperative pain in patients undergoing total hysterectomy [published online ahead of print May 12, 2015]. Pharmacogenomics J. http://dx.doi.org/10.1038/tpj.2015.33.
Effect of catechol-O-methyltransferase-gene (COMT) variants on experimental and acute postoperative pain in 1,000 women undergoing surgery for breast cancer.
exhibit decreased levels of COMT alongside increased levels of catecholamines. Preclinical studies further revealed that elevated levels of epinephrine/norepinephrine resulting from low COMT activity lead to increased pain through activation of β-ARs.
Coming full circle, results from a randomized controlled trial documented that the β-AR antagonist propranolol provides considerable pain relief for patients who have SNPs associated with decreased levels of COMT.
Effect of catechol-O-methyltransferase polymorphism on response to propranolol therapy in chronic musculoskeletal pain: a randomized, double-blind, placebo-controlled, crossover pilot study.
Together, these findings highlight the impact of gene regulation on pain as well as the utility of genetic and protein biomarkers in identifying a subgroup of patients who will benefit from specific therapies.
In a similar fashion, we believe that measurement of alternative GPCR splice variants can be used as a diagnostic tool to provide personalized pain treatment. This procedure is already being done in patients with cancer. In vitro studies examining the role of NK-1R alternative splicing in breast cancer cells revealed that overexpression of the NK-1Rtruncated variant promotes tumorigenesis.
Effect of truncated neurokinin-1 receptor expression changes on the interaction between human breast cancer and bone marrow-derived mesenchymal stem cells.
A complementary clinical study further documented that individuals with overexpression of the NK-1Rtruncated variant were at increased risk for colitis-associated carcinoma, whereas expression levels of the canonical NK1R remained consistent between cases and controls.
Just as the study of alternative splicing is beginning to inform diagnosis and management of patients with cancer, the study of alternative splicing in pain-relevant GPCRs has great potential to advance the current state of clinical care for patients with chronic pain. Additionally, this line of inquiry may lead to the advent of new pain therapies such as IBNtxA, a novel opioid analgesic specifically targeting 6-TM μ-opioid splice variants.
Lu Z, Xu J, Rossi GC, Majumdar S, Pasternak GW, Pan Y-X. Mediation of opioid analgesia by a truncated 6-transmembrane GPCR [published online ahead of print May 26, 2015]. J Clin Invest. http://dx.doi.org/10.1172/JCI81070.
G protein–coupled receptors play a major role in modulating the activity of a chorus of cells involved in the transmission, modulation, and perception of pain. For this reason, GPCRs are the primary target of many pharmacological interventions used in the management of acute and chronic pain. Nonetheless, the use of these medications is limited because of variability in analgesic efficacy and adverse effect profiles. These limitations are partly attributed to genetic differences that influence alternative splicing of pain-relevant GPCRs. The functional importance and implications of the diversity of GPCRs in contributing to the pathophysiology of clinical pain is just beginning to emerge. More research, especially in the clinical arena, is necessary to further investigate the functions of specific GPCR splice variants, as well as the dynamic interactions between multiple variants of the same canonical receptor, within the context of pain. This line of inquiry will evolve our understanding of pain mechanisms and inform the design of new and clinically useful drugs that target specific alternative splice variants altered in a subset of patients.
References
Woolf C.J.
American College of Physicians, American Physiological Society. Pain: moving from symptom control toward mechanism-specific pharmacologic management.
Differential expressions of the alternatively spliced variant mRNAs of the μ opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains.
Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in Alzheimer's disease brains.
COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord.
Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands.
Acute agonist-mediated desensitization of the human α1a-adrenergic receptor is primarily independent of carboxyl terminus regulation: implications for regulation of α1aAR splice variants.
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