Achla Gupta

Hemopressin is an inverse agonist of CB1 cannabinoid receptors.

To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB1 cannabinoid receptors, we used the recently described conformation-state sensitive antibodies and screened a panel of endogenous peptides from rodent brain or adipose tissue. This led to the identification of hemopressin (PVNFKFLSH) as a peptide ligand that selectively binds CB1 cannabinoid receptors. We find that hemopressin is a CB1 receptor-selective antagonist, because it is able to efficiently block signaling by CB1 receptors but not by other members of family A G protein-coupled receptors (including the closely related CB2 receptors). Hemopressin also behaves as an inverse agonist of CB1 receptors, because it is able to block the constitutive activity of these receptors to the same extent as its well characterized antagonist, rimonabant. Finally, we examine the activity of hemopressin in vivo using different models of pain and find that it exhibits antinociceptive effects when administered by either intrathecal, intraplantar, or oral routes, underscoring hemopressins therapeutic potential. These results represent a demonstration of a peptide ligand for CB1 cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB1 receptors.

G protein coupled receptor dimerization: implications in modulating receptor function

Abstract Protein-protein interactions are involved in the regulation of a large number of biological processes. It is well established that a variety of cell surface receptors interact with each other to form dimers, and that this is essential for their activation. Although the existence of G protein coupled receptor dimers was predicted from early pharmacological and biochemical analysis, solid evidence supporting dimerization has come within the past few years following the cloning of G protein coupled receptor cDNAs. Using differential epitope tagging and selective immunoisolation of receptor complexes, dimerization of a number of G protein coupled receptors including members of the rhodopsin, secretin, and metabotropic glutamate receptor families have been reported. More recently fluorescence or bioluminescence resonance energy transfer techniques have been used to examine dimerization of these receptors in live cells. These studies have found that whereas in some cases there is an agonist induced increase in the level of dimers, in others there is a decrease or no change in dimer levels. Several recent studies have also reported the ability of related members of G protein coupled receptors to heterodimerize. These heterodimers exhibit distinct physical and functional properties. Examination of possible sites of interactions between receptors has implicated a role for extracellular, transmembrane and/or C-terminal region in dimerization. The functional consequences of dimerization, explored mainly using mutant receptors, have demonstrated a role in modulating agonist affinity, efficacy, and/or trafficking properties. Thus dimerization appears to be a universal phenomenon that provides an additional mechanism for modulation of receptor function as well as cross-talk between G protein coupled receptors.

Increased abundance of opioid receptor heteromers after chronic morphine administration.

The μ-δ opioid heteromer may be a target for alleviation of chronic pain. Drug-Induced Heteromers Opioid receptors are G protein–coupled receptors and are divided into μ, δ, and κ subtypes. Homomers of μ or δ receptors signal through a Gαi-mediated pathway; however, these receptor subtypes can also form heteromers that signal through Gαz- or β-arrestin2–mediated pathways. Although morphine analgesia is mediated primarily through the μ receptor, δ receptor ligands can potentiate μ receptor–mediated signaling, suggesting that the μ-δ heteromer may also be involved in morphine analgesia. Gupta et al. developed an antibody that selectively recognizes μ-δ heteromers and found that the abundance of the μ-δ heteromer in mice increased with chronic administration of morphine. These increases were detected in regions of the brain that are involved in the modulation of pain transmission. These results suggest that the μ-δ heteromer may be a candidate for therapies to alleviate chronic pain syndromes. The μ and δ types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the μ-δ heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of μ-δ heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the μ-δ heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

Novel endogenous peptide agonists of cannabinoid receptors

Hemopressin (Hp), a 9‐residue α‐hemoglobin‐derived peptide, was previously reported to function as a CB1 cannabinoid receptor antagonist (1). In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N‐terminally extended forms of Hp containing either three (RVD‐Hpa) or two (VD‐Hpa) additional amino acids, as well as a β‐hemoglobin‐derived peptide with sequence similarity to that of hemopressin (VD‐Hpβ). Characterization of the α‐hemoglobin‐derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB1 cannabinoid receptor antagonist, both RVD‐Hpa and VD‐Hpα function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca2+ indicate that these peptides activate a signal transduction pathway distinct from that activated by the endocannabinoid, 2‐arachidonoylglycerol, or the classic CB1 agonist, Hu‐210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB1 receptor is involved in the integration of signals from both lipid‐ and peptide‐derived signaling molecules.—Gomes, I., Grushko, J. S., Golebiewska, U., Hoogendoorn, S., Gupta, A., Heimann, A. S., Ferro, E. S., Scarlata, S., Fricker, L. D., Devi, L. A. Novel endogenous peptide agonists of cannabinoid receptors. FASEB J. 23, 3020–3029 (2009). www.fasebj.org

Conformation State-sensitive Antibodies to G-protein-coupled Receptors

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of μ-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of μ receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize μ receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native μ receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other Gαi-coupled (δ-opioid, CB1 cannabinoid, α2A-adrenergic) as well as Gαs-(β2-adrenergic) and Gαq-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.

Cell surface targeting of μ-δ opioid receptor heterodimers by RTP4

μ opioid receptors are G protein–coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that μ-δ opioid heterodimers have a pharmacologic profile distinct from those of the μ or δ homodimers. Because the heterodimers exhibit distinct signaling properties, the protein and mechanism regulating their levels have significant effects on morphine-mediated physiology. We report the characterization of RTP4, a Golgi chaperone, as a regulator of the levels of heterodimers at the cell surface. We show that the association with RTP4 protects μ-δ receptors from ubiquitination and degradation. This leads to increases in surface heterodimer levels, thereby affecting signaling. Thus, the oligomeric organization of opioid receptors is controlled by RTP4, and this governs their membrane targeting and functional activity. This work is the first report of the identification of a chaperone involved in the regulation of the biogenesis of a family A GPCR heterodimer. The identification of such factors as RTP4 controlling dimerization will provide insight into the regulation of heterodimers in vivo. This has implications in the modulation of pharmacology of their endogenous ligands, and in the development of drugs with specific therapeutic effects.

AT1R–CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II

The mechanism of G protein‐coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero‐oligomers facilitates signal integration of different receptor types, cross‐talk between Gαi‐ and Gαq‐coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi‐coupled type I cannabinoid receptor (CB1R) and the Gαq‐coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co‐immunoprecipitation and resonance energy transfer assays, as well as receptor‐ and heteromer‐selective antibodies, we show that AT1R and CB1R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol‐administered rats in which CB1R is upregulated. We found a significant upregulation of AT1R–CB1R heteromers and enhancement of angiotensin II‐mediated signalling, as compared with cells from control animals. Moreover, blocking CB1R activity prevented angiotensin II‐mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer‐dependent signal integration in pathology.

Identification of a μ-δ opioid receptor heteromer-biased agonist with antinociceptive activity

G protein-coupled receptors play a pivotal role in many physiological signaling pathways. Mounting evidence suggests that G protein-coupled receptors, including opioid receptors, form dimers, and dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological role of dimerization in vivo has not been well-explored because of the lack of tools to study these dimers in endogenous systems. To address this problem, we previously generated antibodies to μ-δ opioid receptor (μOR-δOR) dimers and used them to study the pharmacology and signaling by this heteromer. We also showed that the heteromer exhibits restricted distribution in the brain and that its abundance is increased in response to chronic morphine administration. Thus, the μOR-δOR heteromer represents a potentially unique target for the development of therapeutics to treat pain. Here, we report the identification of compounds targeting μOR-δOR heteromers through high-throughput screening of a small-molecule library. These compounds exhibit activity in μOR-δOR cells but not μOR or δOR cells alone. Among them, CYM51010 was found to be a μOR-δOR–biased ligand, because its activity is blocked by the μOR-δOR heteromer antibody. Notably, systemic administration of CYM51010 induced antinociceptive activity similar to morphine, and chronic administration of CYM51010 resulted in lesser antinociceptive tolerance compared with morphine. Taken together, these results suggest that CYM51010, a μOR-δOR–biased ligand, could serve as a scaffold for the development of a unique type (heteromer-biased) of drug that is more potent and without the severe side effects associated with conventional clinical opioids.

Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.

β2-Adrenergic receptor signaling in the cardiac myocyte is modulated by interactions with CXCR4.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α and its G-protein coupled receptor, CXCR4. Stromal cell-derived factor-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective β-adrenergic receptor (βAR) agonist, isoproterenol. As a result of the importance of β-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of βAR signaling. Our studies demonstrate activation of CXCR4 by stromal cell-derived factor-1 leads to a decrease in βAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of βAR downstream targets is β2AR-dependent. We demonstrated a physical interaction between CXCR4 and β2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-β2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-β2AR complex could represent a hitherto unknown target for therapeutic intervention.