A single mutation in the 5-HT4 receptor (5-HT4-R D100(3.32)A) generates a Gs-coupled receptor activated exclusively by synthetic ligands (RASSL)

To better understand G-protein-coupled receptor (GPCRs) signaling, cellular and animal physiology, as well as gene therapy, a new tool has recently been proposed. It consists of GPCR mutants that are insensitive to endogenous ligands but sensitive to synthetic ligands. These GPCRs are called receptor activated solely by synthetic ligands (RASSL). Only two examples of such engineered receptors have been described so far: one G(i)-coupled (opioid receptors) and one G(s)-coupled (beta(2)-adrenergic receptors). Here, we describe the first RASSL related to serotonin receptors (D100(3.32)A G(s)-coupled 5-HT(4) receptor or 5-HT(4)-RASSL). 5-HT(4)-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still responds to synthetic ligands. These ligands have affinities in the range of nanomolar concentrations for the mutant receptor and exhibit full efficacy. More interestingly, two synthetic ligands behave as antagonists on the wild type but as agonists on the 5-HT(4)-RASSL.     

Engineering the melanocortin-4 receptor to control constitutive and ligand-mediated G(S) signaling in vivo

The molecular and functional diversity of G protein-coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein-mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of G(s) signaling in vivo. We used naturally occurring human mutations to develop two G(s)-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our G(s)-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone alpha-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the G(s) pathway in vivo. These RASSLs can be used to activate G(s) signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.     

A G(q/11)-coupled mutant histamine H(1) receptor F435A activated solely by synthetic ligands (RASSL)

Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study Galpha(i) signaling in vivo (1, 2). Also, Galpha(s)-coupled G protein-coupled receptors have been engineered to generate Galpha(s)-coupled RASSLs (3, 4). In this study, we exploited the differences in binding pockets between different classes of H(1) receptor agonists and identified the first Galpha(q/11)-coupled RASSL. The mutant human H(1) receptor F435A (6.55) combines a strongly decreased affinity (25-fold) and potency for the endogenous ligand histamine (200-fold) with improved affinities (54-fold) and potencies (2600-fold) for 2-phenylhistamines, a synthetic class of H(1) receptor agonists. Molecular dynamics simulations provided a mechanism for distinct agonist binding to both wild-type and F435A mutant H(1) receptors.     

Chimeric G Proteins Allow a High-Throughput Signaling Assay of Gi-Coupled Receptors

G-protein-coupled receptors are a major target for potential therapeutics; yet, a large number of these receptors couple to the Gi pathway, generating signals that are difficult to detect. We have combined chimeric G proteins, automated sample handling, and simultaneous 96-well fluorometric imaging to develop a high-throughput assay system for Gi signaling. The chimeric G proteins alter receptor coupling so that signaling can occur through Gq and result in mobilization of intracellular calcium stores. An automated signaling assay device, the fluorometric imaging plate reader (FLIPR), can simultaneously measure this response in real time in 96-well microplates, allowing two people to process more than 10,000 points per day. We used the chimeric G protein/FLIPR system to characterize signaling by the Gi-coupled human opioid receptors. We show that the mu, delta, and kappa opioid receptors and the related nociceptin receptor, ORL1, each couple to Galphaqi5, Galphaqo5, and Galpha16 (Galphaqi5 and Galphaqo5 refer to Galphaq proteins containing the five carboxyl-terminal amino acids from Galphai and Galphao, respectively) and that different receptor/G protein combinations show different levels of maximal activation. We tested 31 opioid ligands for agonist activity at the opioid receptors (124 ligand-receptor combinations); all 31 activated at least one receptor type, and several activated multiple receptors with differing potencies. This high-throughput assay could be useful for dissecting the complex ligand-receptor relationships that are common in nature.     

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor

G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT(4b) receptor, a GPCR with high constitutive G(s) signaling and strong ligand-induced G-protein activation of the G(s) and G(s/q) pathways. The first receptor in this series, 5-HT(4)-D(100)A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G(s) signaling, but only a few (e.g., zacopride) also induced signaling via the G(q) pathway. Zacopride-induced G(q) signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT(2C) receptor. Additional point mutations (D(66)A and D(66)N) blocked constitutive G(s) signaling and lowered ligand-induced G(q) signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT(1A) conferred ligand-mediated G(i) signaling. This G(i)-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G(s) or G(q) pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.     

The role of mu opioid receptor desensitization and endocytosis in morphine tolerance and dependence

Following activation, most G protein coupled receptors undergo regulation by a cascade of events that promote receptor desensitization and endocytosis. Following endocytosis, receptors can then be recycled to the plasma membrane, retained in an intracellular compartment, or targeted for degradation. For receptors that are recycled, like the mu opioid receptor (MOR), endocytosis serves as the first step toward resensitizing receptors. For receptors that are degraded, endocytosis serves as the first step toward receptor downregulation. Thus, for receptors like the MOR, the desensitization-endocytosis-resensitization cycle serves as a rapid and dynamic means to titrate signaling through the receptor. However, not all agonist ligands at the MOR promote the same degree of receptor desensitization and endocytosis. For example, the endogenous peptide ligands at the MOR induce rapid desensitization, endocytosis, and recycling. By contrast, morphine induces only weak or partial desensitization and little to no endocytosis. As a consequence, signal transduction promoted by morphine is less dynamic than that induced by endogenous ligands as well as other opioid agonists that promote endocytosis. The resulting imbalance of desensitization-endocytosis-resensitization has at least two consequences: (1) in cell types where morphine induces desensitization but not endocytosis and/or resensitization, desensitization is protracted; (2) in cell types where morphine induces neither desensitization nor endocytosis, prolonged signaling through the receptor leads to multiple cellular adaptations downstream of receptor-G protein coupling. Both protracted desensitization and adaptive cellular changes probably contribute to the pronounced in vivo tolerance and dependence that occur with chronic morphine treatment. As a consequence, facilitating receptor endocytosis, using either genetic or pharmacological approaches, can restore the balance of signaling through the receptor and affect the development of tolerance and dependence.     

Interactions between opioid and chemokine receptors: heterologous desensitization

The opioid and chemokine receptors are both members of the seven transmembrane G protein-coupled receptor (GPCR) superfamily. Desensitization is believed to be a major element of the regulation of the function of these receptors, and recent findings suggest that both agonist-dependent (homologous) desensitization and heterologous desensitization can control receptor activity. The cross-desensitization between opioid and chemokine receptors has significant implications for our understanding of both the regulation of leukocyte trafficking, as well as the regulation of chemokine receptor function in inflammatory disease states. We also review findings which suggest that pro-inflammatory chemokine receptor-induced heterologous desensitization of opioid receptors has important implications for the regulation of opioid receptor function in the nervous system.     

Opioid tolerance-in search of the holy grail

Tolerance, one of several behavioral adaptations to prolonged opioid treatment, has long been explained by desensitization of opioid receptor signaling and loss of surface receptors. However, recent evidence presents an alternative hypothesis, suggesting that receptor internalization could in fact reduce tolerance in vivo.     

Molecular and cellular mechanisms of the age-dependency of opioid analgesia and tolerance

ABSTRACT: The age-dependency of opioid analgesia and tolerance has been noticed in both clinical observation and laboratory studies. Evidence shows that many molecular and cellular events that play essential roles in opioid analgesia and tolerance are actually age-dependent. For example, the expression and functions of endogenous opioid peptides, multiple types of opioid receptors, G protein subunits that couple to opioid receptors, and regulators of G protein signaling (RGS proteins) change with development and age. Other signaling systems that are critical to opioid tolerance development, such as N-methyl-D-aspartic acid (NMDA) receptors, also undergo age-related changes. It is plausible that the age-dependent expression and functions of molecules within and related to the opioid signaling pathways, as well as age-dependent cellular activity such as agonist-induced opioid receptor internalization and desensitization, eventually lead to significant age-dependent changes in opioid analgesia and tolerance development.     

A role for the distal carboxyl tails in generating the novel pharmacology and G protein activation profile of mu and delta opioid receptor hetero-oligomers

Opioid receptor pharmacology in vivo has predicted a greater number of receptor subtypes than explained by the profiles of the three cloned opioid receptors, and the functional dependence of the receptors on each other shown in gene-deleted animal models remains unexplained. One mechanism for such findings is the generation of novel signaling complexes by receptor hetero-oligomerization, which we previously showed results in significantly different pharmacology for mu and delta receptor hetero-oligomers compared with the individual receptors. In the present study, we show that deltorphin-II is a fully functional agonist of the mu-delta heteromer, which induced desensitization and inhibited adenylyl cyclase through a pertussis toxin-insensitive G protein. Activation of the mu-delta receptor heteromer resulted in preferential activation of Galpha(z), illustrated by incorporation of GTPgamma(35)S, whereas activation of the individually expressed mu and delta receptors preferentially activated Galpha(i). The unique pharmacology of the mu-delta heteromer was dependent on the reciprocal involvement of the distal carboxyl tails of both receptors, so that truncation of the distal mu receptor carboxyl tail modified the delta-selective ligand-binding pocket, and truncation of the delta receptor distal carboxyl tail modified the mu-selective binding pocket. The distal carboxyl tails of both receptors also had a significant role in receptor interaction, as evidenced by the reduced ability to co-immunoprecipitate when the carboxyl tails were truncated. The interaction between mu and delta receptors occurred constitutively when the receptors were co-expressed, but did not occur when receptor expression was temporally separated, indicating that the hetero-oligomers were generated by a co-translational mechanism.     

Expression of a Gi-coupled receptor in the heart causes impaired Ca2+ handling, myofilament injury, and dilated cardiomyopathy

Increased signaling by G(i)-coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted G(i)-coupled receptor (Ro1). Activation of G(i) signaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff-perfused hearts. However, acute termination of Ro1 signaling with the antagonist nor-binaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiation of Ro1 expression, there was a 40% reduction in the abundance of the sarcoplasmic reticulum Ca(2+)-ATPase (P < 0.05); thereafter, there was progressive impairment of both Ca(2+) handling and force development assessed with ventricular trabeculae. Six weeks after initiation of Ro1 expression, systolic Ca(2+) concentration was reduced to 0.61 +/- 0.08 vs. 0.91 +/- 0.07 microM for control (n = 6-8; P < 0.05), diastolic Ca(2+) concentration was elevated to 0.41 +/- 0.07 vs. 0.23 +/- 0.06 microM for control (n = 6-8; P < 0.01), and the decline phase of the Ca(2+) transient (time from peak to 50% decline) was slowed to 0.25 +/- 0.02 s vs. 0.13 +/- 0.02 s for control (n = 6-8; P < 0.01). Early after initiation of Ro1 expression, there was a ninefold elevation of matrix metalloproteinase-2 (P < 0.01), which is known to cause myofilament injury. Consistent with this, 6 wk after initiation of Ro1 expression, Ca(2+)-saturated myofilament force in skinned trabeculae was reduced to 21 +/- 2 vs. 38 +/- 0.1 mN/mm(2) for controls (n = 3; P < 0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure in which increased activity of G(i)-coupled receptors leads to impaired Ca(2+) handling and myofilament injury, contributing to impaired ventricular pump function and heart failure.     

Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.     

Abnormal contraction caused by expression of G(i)-coupled receptor in transgenic model of dilated cardiomyopathy

Although increased G(i) signaling has been associated with dilated cardiomyopathy in humans, its role is not clear. Our goal was to determine the effects of chronically increased G(i) signaling on myocardial function. We studied transgenic mice that expressed a G(i)-coupled receptor (Ro1) that was targeted to the heart and regulated by a tetracycline-controlled expression system. Ro1 expression for 8 wk resulted in abnormal contractions of right ventricular muscle strips in vitro. Ro1 expression reduced myocardial force by >60% (from 35 +/- 3 to 13 +/- 2 mN/mm(2), P < 0.001). Nevertheless, sensitivity to extracellular Ca(2+) was enhanced. The extracellular [Ca(2+)] resulting in half-maximal force was lower with Ro1 expression compared with control (0.41 +/- 0.05 vs. 0.88 +/- 0.05 mM, P < 0.001). Ro1 expression slowed both contraction and relaxation kinetics, increasing the twitch time to peak (143 +/- 6 vs. 100 +/- 4 ms in control, P < 0.001) and the time to half relaxation (124 +/- 6 vs. 75 +/- 6 ms in control, P < 0.001). Increased pacing frequency increased contractile force threefold in control myocardium (P < 0.001) but caused no increase of force in Ro1-expressing myocardium. When stimulation was interrupted with rests, postrest force increased in control myocardium, but there was postrest decay of force in Ro1-expressing myocardium. These results suggest that defects in contractility mediated by G(i) signaling may contribute to the development of dilated cardiomyopathy.     

Beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization

Extracellular calcium rapidly controls PTH secretion through binding to the G protein-coupled calcium-sensing receptor (CASR) expressed in parathyroid glands. Very little is known about the regulatory proteins involved in desensitization of CASR. G protein receptor kinases (GRK) and beta-arrestins are important regulators of agonist-dependent desensitization of G protein-coupled receptors. In the present study, we investigated their role in mediating agonist-dependent desensitization of CASR. In heterologous cell culture models, we found that the transfection of GRK4 inhibits CASR signaling by enhancing receptor phosphorylation and beta-arrestin translocation to the CASR. In contrast, we found that overexpression of GRK2 desensitizes CASR by classical mechanisms as well as through phosphorylation-independent mechanisms involving disruption of Galphaq signaling. In addition, we observed lower circulating PTH levels and an attenuated increase in serum PTH after hypocalcemic stimulation in beta-arrestin2 null mice, suggesting a functional role of beta-arrestin2-dependent desensitization pathways in regulating CASR function in vivo. We conclude that GRKs and beta-arrestins play key roles in regulating CASR responsiveness in parathyroid glands.     

Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.     

The absence of a direct correlation between the loss of [D-Ala2, MePhe4,Gly5-ol]Enkephalin inhibition of adenylyl cyclase activity and agonist-induced mu-opioid receptor phosphorylation

Chronic activation of the mu-opioid receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the receptor. It has been suggested that opioid receptor phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO) induced a time- and concentration-dependent phosphorylation of the receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the receptor, but is not involved in DAMGO-induced MOR1TAG phosphorylation. The rapid rate of receptor phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between receptor phosphorylation and the loss of response was further demonstrated when receptor phosphorylation was increased by either calyculin A or overexpression of the G-protein receptor kinases. Calyculin A increased the magnitude of MOR1TAG phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when mu- and delta-opioid (DOR1TAG) receptors were expressed in the same system, overexpression of beta-adrenergic receptor kinase 2 elevated agonist-induced phosphorylation for both receptors. However, in the same cell lines under the same conditions, overexpression of beta-adrenergic receptor kinase 2 and beta-arrestin 2 accelerated the rate of DPDPE- but not DAMGO-induced receptor desensitization. Thus, these data show that phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the receptor.     

Reconstitution reveals additional roles for N- and C-terminal domains of g(alpha) in muscarinic receptor coupling

The molecular basis for selectivity of M1 and M2 muscarinic receptor coupling to heterotrimeric G proteins has been studied using receptors expressed in Sf9 cell membranes and reconstituted with purified chimeric G(alpha) subunits containing different regions of Gi1alpha and Gq(alpha). The abilities of G protein heterotrimers containing chimeric alpha subunits to stabilize the high-affinity state of the receptors for agonist and to undergo receptor stimulated guanine nucleotide exchange was compared with G protein heterotrimers containing either native Gi1alpha or Gq(alpha). The data confirm the importance of the proper context of the C-terminus of Galpha by demonstrating that the C-terminus of Gi1alpha, when placed in the context of Gq(alpha), prevents coupling to muscarinic M1 receptors, while the C-terminus of Gq(alpha), when placed in the context of Gi1alpha, prevents coupling to muscarinic M2 receptors. However, C-terminal amino acids of Gq(alpha) placed in the context of Gi1alpha were not sufficient to allow M1 receptor coupling, nor were C-terminal amino acids of Gi1alpha placed in the context of Gq(alpha) sufficient for M2 receptor coupling. The unique six amino acid N-terminal extension of Gq(alpha) when added to the N-terminus of Gi1alpha neither prevented M2 receptor coupling nor permitted M1 receptor coupling. A Gi1alpha-based chimera containing both N- and C-terminal regions of Gq(alpha) gained the ability to productively couple M1 receptors suggesting that the proper context of both N- and C-termini is required for muscarinic receptor coupling.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

G protein-coupled receptor crosstalk and signaling in hematopoietic stem and progenitor cells

A variety of G protein-coupled receptors (GPCRs) is expressed in hematopoietic stem and progenitor cells (HPCs), including the chemokine receptor CXCR4, the leukotriene receptor CysLT1, the sphingosine 1-phosphate receptor S1P1, the cannabinoid receptor CB2, and the complement receptor C3aR. While the role of CXCR4 in stem cell homing is largely established, the function of the other GPCRs expressed in HPCs is only partially understood. CXCR4 and CysLT1 inhibit their own activation after ligand binding (homologous desensitization). Stimulation of S1P1 or C3aR has been shown to activate CXCR4 in HPCs that may sensitize CXCR4-dependent stem cell homing. In contrast, activation of CXCR4 results in a loss of CysLT1 function, which is most likely mediated by protein kinase C (PKC) signaling (heterologous desensitization) and could explain the ineffectiveness of CysLT1 antagonists to mobilize HPCs in vivo. Further characterization of GPCR crosstalk will allow a better understanding of HPC trafficking.     

Engineered G protein coupled receptors reveal independent regulation of internalization,desensitization and acute signaling

Background  

The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of G_i-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization.