VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.     

Effect of the A118G polymorphism on binding affinity, potency and agonist-mediated endocytosis, desensitization, and resensitization of the human mu-opioid receptor

The most prevalent single-nucleotide polymorphism (SNP) A118G in the human mu-opioid receptor gene predicts an amino acid change from an asparagine residue to an aspartatic residue in amino acid position 40. This N40D mutation, which has been implicated in the development of opioid addiction, was previously reported to result in an increased beta-endorphin binding affinity and a decreased potency of morphine-6-glucuronide. Therefore, in the present study we have investigated whether this mutation might affect the binding affinity, potency, and/or the agonist-induced desensitization, internalization and resensitization of the human mu-opioid receptor stably expressed in human embryonic kidney 293 cells. With the exception of a reduced expression level of N40D compared to human mu-opioid receptor (hMOR) in HEK293 cells, our analyses revealed no marked functional differences between N40D and wild-type receptor. Morphine, morphine-6-glucuronide and beta-endorphin revealed similar binding affinities and potencies for both receptors. Both the N40D-variant receptor and hMOR exhibited robust receptor internalization in the presence of the opioid peptide [d-Ala(2),N-MePhe(4),Glyol(5)]enkephalin (DAMGO) and beta-endorphin but not in response to morphine or morphine-6-glucuronide. After prolonged treatment with morphine, morphine-6-glucuronide or beta-endorphin both receptors showed similiar desensitization time courses. In addition, the receptor resensitization rates were nearly identical for both receptor types.     

Relationship between the G protein signaling and homologous desensitization of G protein-coupled receptors

Signaling and desensitization of G protein-coupled receptor are intimately related, and measuring them separately requires certain parameters that represent desensitization independently of signaling. In this study, we tested whether desensitization requires signaling in three different receptors, beta2-adrenergic receptor (beta2AR) in S49 lymphoma cells, alpha-factor pheromone receptor (Ste2p) in Saccharomyces cerevisiae LM102 cells, and dopamine D3 receptor (D3R) in HEK-293 cells. Agonist-induced beta-arrestin translocation to the plasma membrane or receptor sequestration was measured to estimate homologous desensitization. To separate the signaling and desensitization of beta2AR, which mediates stimulation of adenylyl cyclase, S49 lymphoma cys- cells that lack the alpha subunit of Gs were used. Stimulation of beta2AR in these cells failed to increase intracellular cAMP, but beta-arrestin translocation still occurred, suggesting that feedback from beta2AR signaling is not required for homologous desensitization to occur. Agonist-induced sequestration of the yeast Ste2p-L236R, which showed reduced signaling through G protein, was not different from that of wildtype Ste2p, suggesting that the receptor signaling and sequestration are not directly linked cellular events. Both G protein coupling and D3R signaling, measured as inhibition of cAMP production, were greatly enhanced by co-expression of exogenous alpha subunit of Go (Goalpha) or adenylyl cyclase type 5 (AC5), respectively. However, agonist-induced beta-arrestin translocation, receptor phosphorylation, and sequestration were not affected by co-expression of Galphao and AC5, suggesting that the extent of signaling does not determine desensitization intensity. Taken together, our results consistently suggest that G protein signaling and homologous desensitization are independent cellular processes.     

Morphine-induced mu-opioid receptor rapid desensitization is independent of receptor phosphorylation and beta-arrestins

Receptor desensitization involving receptor phosphorylation and subsequent betaArrestin (betaArr) recruitment has been implicated in the tolerance development mediated by mu-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and betaArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca(2+) ([Ca(2+)](i))release to monitor receptor activation, as predicted, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation- and betaArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from betaArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from betaArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca(2+)](i) release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser(375) residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking betaArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of betaArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of betaArr.     

Selective interactions between helix VIII of the human mu-opioid receptors and the C terminus of periplakin disrupt G protein activation

Analysis of interactions between the C-terminal tail of the MOP-1 and MOP-1A variants of the human mu-opioid receptor with proteins derived from a human brain cDNA library resulted in identification of the actin and intermediate filament-binding protein periplakin. Mapping of this interaction indicated that the predicted fourth intracellular loop/helix VIII of the receptor interacts with the C-terminal rod and linker region of periplakin. Periplakin is widely expressed in the central nervous system of both man and rat and demonstrated an overlapping but not identical distribution with mu-opioid (MOP) receptors. Co-expression of periplakin with MOP-1 or a MOP-1-eYFP fusion construct in HEK293 cells did not interfere with agonist-mediated internalization of the receptor. When co-expressed with a MOP-1-Gi1 alpha fusion protein periplakin significantly reduced the capacity of the agonist to stimulate binding of 35S-labeled guanosine 5'-3-O-(thio)triphosphate ([35S]GTP gamma S) to the receptor-associated G protein. By contrast, periplakin did not interfere with agonist-stimulation of [35S]GTP gamma S binding to either an alpha 2A-adrenoreceptor-Gi1 alpha fusion protein or a beta2-adrenoreceptor-Gs alpha fusion protein, indicating its selectivity of function. This represents the first example of an opioid receptor-interacting protein that functions to disrupt agonist-mediated G protein activation.     

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.     

Modeling activation and desensitization of G-protein coupled receptors provides insight into ligand efficacy.

Signaling through G-protein coupled receptors is one of the most prevalent and important methods of transmitting information to the inside of cells. Many mathematical models have been proposed to describe this type of signal transduction, and the ternary complex (ligand/receptor/G-protein) model and its derivatives are among the most widely accepted. Current versions of these equilibrium models include both active (i.e. signaling) and inactive conformations of the receptor, but do not include the dynamics of G-protein activation or receptor desensitization. Yet understanding how these dynamic events effect response behavior is crucial to determining ligand efficacy. We developed a mathematical model for G-protein coupled receptor signaling that includes G-protein activation and receptor desensitization, and used it to predict how activation and desensitization would change if either the conformational selectivity (the effect of ligand binding on the distribution of active and inactive receptor states) or the desensitization rate constant was ligand-specific. In addition, the model was used to explore the implications of measuring responses far downstream from G-protein activation. By comparing the experimental data from the beta(2)-adrenergic, micro-opioid, D(1)dopamine, and neutrophil N -formyl peptide receptors with the predictions of our model, we found that the conformational selectivity is the predominant factor in determining the amounts of activation and desensitization caused by a particular ligand.     

Agonist-specific down regulation of mu-opioid receptors: Different cellular pathways are activated by different opioid agonists

Opioid agonists are known to induce down regulation of opioid receptors through the classical pathway that involves phosphorylation, clathrin-dependent endocytosis and lysosomal/endosomal degradation of the internalized receptors. As expected, exposure of mu-opioid receptor (MOR)-transfected HEK-293 cells to either DAMGO (a specific mu-opioid agonist) or etorphine (a wide spectrum opioid agonist) resulted in down regulation of the receptors that was blocked by the kinase inhibitor staurosporine, by hypertonic sucrose and by the lysosomal and proteasomal inhibitors chloroquine and lactacystin. High concentration of etorphine, but not of DAMGO, induced an additional process of down regulation that was resistant to staurosporine, to hypertonic sucrose and to chloroquine-lactacystin. Etorphine, but not DAMGO, also induced down regulation of mu-opioid receptors in isolated membranes of HEK cells. This membrane-delimited down regulation was blocked by selective inhibitors of protease enzymes, suggesting the involvement of membranous serine- and amino-peptidases. This membranous down regulation of opioid receptors was dependent on the concentration of etorphine and was blocked by the opioid antagonist naloxone. Etorphine induced similar down regulation in membranes of HEK-293 cells transfected with delta-opioid receptors (DOR) as well in membranes of cells that endogenously express opioid receptors. This agonist-specific membrane-delimited regulatory process appears to be physiologically relevant and should be taken into account when studying long term effects of opioid drugs.     

Untangling ligand induced activation and desensitization of G-protein-coupled receptors

Long-term treatment with a drug to a G-protein-coupled receptor (GPCR) often leads to receptor-mediated desensitization, limiting the therapeutic lifetime of the drug. To better understand how this therapeutic window might be controlled, we created a mechanistic Monte Carlo model of the early steps in GPCR signaling and desensitization. Using this model we found that the rates of G-protein activation and receptor phosphorylation can be partially decoupled by varying the drug-receptor dissociation rate constant, k(off), and the drug's efficacy, alpha. The maximum ratio of G-protein activation to receptor phosphorylation (GARP) was found for drugs with an intermediate k(off) value and small alpha-value. Changes to the cellular environment, such as changes in the diffusivity of membrane molecules and the G-protein inactivation rate constant, affected the GARP value of a drug but did not change the characteristic shape of the GARP curve. These model results are examined in light of experimental data for a number of GPCRs and are found to be in good agreement, lending support to the idea that the desensitization properties of a drug might be tailored to suit a specific application.     

Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors

The actions of four tachykinins on inhibition and desensitization of the M-current of bullfrog sympathetic neurons have been characterized. Radioligand binding parameters of the tachykinins were determined at a neurokinin receptor in a heterologous expression system. The correlation between binding, signaling and receptor regulation was investigated. A correlation between receptor binding and signaling was found between the peptides; however, their ability to produce desensitization was not correlated with binding and signaling. These results show that the ability of a tachykinin peptide to induce signal activation is not indicative of its ability to induce receptor regulation.     

Calcium-dependent,slow desensitization distinguishes different types of glutamate receptors

1. L-Glutamate, the most likely transmitter of rapid excitatory synaptic interactions in the brain and spinal cord, is a potent neurotoxin. Mechanisms that terminate the action of glutamate are, therefore, likely to be important for maintaining the integrity of glutaminoceptive neurons. In this study, we show that glutamate currents evoked in voltage-clamped chick motoneurons fade during prolonged or repeated application of glutamate by pressure ejection from nearby pipettes. 2. The magnitude of the decline depends on the Ca2+/Mg2+ ratio in the extracellular medium. With Ca2+ = 10.0 mM and no added Mg, the steady-state glutamate current amounted to 50% of the initial value. 3. Single-channel measurements indicate that the fade is due to receptor desensitization rather than to agonist-induced channel blockade, as the mean channel open time within bursts is independent of the agonist concentration. 4. Application of more selective agonists showed that Ca2+-dependent slow desensitization involved only G1 (NMDA) receptors. G2 responses (activated by kainate and quisqualate) did not exhibit this slow phase of desensitization under the same conditions.     

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full-length RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptor-mediated endocytosis.     

A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.     

A novel, generic and effective method for the rapid purification of G protein-coupled receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.     

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.     

The glucagon-like peptide-2 receptor C terminus modulates beta-arrestin-2 association but is dispensable for ligand-induced desensitization, endocytosis, and G-protein-dependent effector activation

Classic models of receptor desensitization and internalization have been largely based on the behavior of Family A G-protein-coupled receptors (GPCRs). The glucagon-like peptide-2 receptor (GLP-2R) is a member of the Family B glucagon-secretin GPCR family, which exhibit significant sequence and structural differences from the Family A receptors in their intracellular and extracellular domains. To identify structural motifs that regulate GLP-2R signaling and cell surface receptor expression, we analyzed the functional properties of a series of mutant GLP-2Rs. The majority of the C-terminal receptor tail was dispensable for GLP-2-induced cAMP accumulation, ERK1/2 activation, and endocytosis in transfected cells. However, progressive truncation of the C terminus reduced cell surface receptor expression, altered agonist-induced GLP-2R trafficking, and abrogated protein kinase A-mediated heterologous receptor desensitization. Elimination of the distal 21 amino acids of the receptor was sufficient to promote constitutive receptor internalization and prevent agonist-induced recruitment of beta-arrestin-2. Site-directed mutagenesis identified specific amino acid residues within the distal GLP-2R C terminus that mediate the stable association with beta-arrestin-2. Surprisingly, although the truncated mutant receptors failed to interact with beta-arrestin-2, they underwent homologous desensitization and subsequent resensitization with kinetics similar to that observed with the wild-type GLP-2R. Our data suggest that, although the GLP-2R C terminus is not required for coupling to cellular machinery regulating signaling or desensitization, it may serve as a sorting signal for intracellular trafficking. Taken together with the previously demonstrated clathrin and dynamin-independent, lipid-raft-dependent pathways for internalization, our data suggest that GLP-2 receptor signaling has evolved unique structural and functional mechanisms for control of receptor trafficking, desensitization, and resensitization.     

Insulin and insulin-like growth factor I receptors utilize different G protein signaling components

We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.     

Characterization of M2 muscarinic receptor activation of different G protein subtypes

The M2 muscarinic acetylcholine receptor (mAChR) expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system formed active functional complexes with coexpressed Gi as well as with Go proteins, while no complexes could be detected with internal G proteins. Comparison of the abilities of different muscarinic agonists and partial agonists to increase [35S]GTPgammaS binding revealed no significant differences between M2/Gi and M2/Go complexes neither with respect to affinities nor efficacies of the ligands studied. Coexpression with either G protein caused constitutive activity of the receptor amounting up to 66% of stimulable [35S]GTPgammaS binding. Muscarinic antagonists, like atropine, scopolamine and N-methylscopolamine, behaved as inverse antagonists with potencies in good agreement with their binding affinities to the receptor. The results implicate that the functional reconstitution of M2 muscarinic receptor with either Gi or Go proteins in insect cells provides a valuable tool for screening of potencies as well as efficacies of agonists, partial agonists and inverse agonists at this receptor.     

Triggered exocytosis and endocytosis have different requirements for calcium and nucleotides in permeabilized bovine chromaffin cells

The intracellular requirements for membrane recapture in permeabilized chromaffin cells were compared to the requirements for exocytosis from the same cells.In permeabilized bovine chromaffin cells, calcium-driven exocytosis also triggers, with a short delay, uptake of extracellular horseradish peroxidase (HRP). This internalized HRP remains compartmentalized within the cell and migrates to a low density band on a Percoll gradient which is distinct from the heavier chromaffin granules.The amount of horseradish peroxidase internalized is similar in intact and leaky cells and is approximately equivalent to the volumes secreted. Endocytosis in both preparations is blocked by botulinum toxin, operates in a collapsed membrane potential, and is inhibited by low temperature. In permeabilized cells, exocytosis and coupled endocytosis are activated by the same concentrations of Ca²⁺ and MgATP. Although secretion requires Ca²⁺ and MgATP, once exocytosis has occurred the subsequent endocytosis can proceed in the virtual absence of Ca²⁺ or MgATP, and is largely unaffected by a variety of nucleotide triphosphates (including nonhydrolyzable analogues), and cyclic nucleotides.These data suggest that endocytosis can proceed, once exocytosis has been triggered, under conditions that are quite different from those necessary to support exocytosis, and that the specific requirements for Ca²⁺ and MgATP in secretion are for the exocytotic limb of the secretory cycle rather than for the associated endocytotic pathway.We are grateful to Mr. John Gibbs for excellent technical assistance, and to the Medical Research Council (UK) for financial support.