Receptors In Human Embryonic Kidney Research Articles

B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin) Is an Inactivation-Resistant Agonist of the Bradykinin B2Receptor Derived from the Peptide Antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-bradykinin): Pharmacologic Profile and Effective Induction of Receptor Degradation

The bradykinin B(2) receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 (d-Arg-[Hyp(3),Igl(5),D-Igl(7),Oic(8)]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B(2) receptor and insurmountable at the rabbit B(2) receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (D-Arg-[Hyp(3),Igl(5),Oic(7),D-Igl(8)]-bradykinin; inverted sequence Oic(7), D-Igl(8)) and B-9972 (D-Arg-[Hyp(3),Igl(5),Oic(7),Igl(8)]-bradykinin); they are low- and high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B(2) receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B(2) receptors: conjugate of the B(2) receptor with green fluorescent protein (B(2)R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B(2) receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B(2) receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B(2)R-GFP desensitization ([(3)H]bradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B(2) receptors by the agonists. The results illustrate the agonist-antagonist transition in B(2) receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist.

Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABAA receptor‐channel kinetic behavior

GABAA receptor subtypes comprising the α1 and α3 subunits change with development and have a specific anatomical localization in the adult brain. These receptor subtypes have been previously demonstrated to greatly differ in deactivation kinetics but the underlying gating mechanisms have not been fully elucidated. Therefore, we expressed rat α1β2γ2 and α3β2γ2 receptors in human embryonic kidney 293 cells and recorded current responses to ultrafast GABA applications at macroscopic and single-channel levels. We found that the slow deactivation of α3β2γ2-mediated currents is associated with a relatively small rate and extent of apparent desensitization. In contrast, responses mediated by α1β2γ2 receptors had faster deactivation and stronger desensitization. α3β2γ2 receptors had faster recovery in the paired-pulse agonist applications than α1β2γ2 channels. The onset of currents mediated by α3β2γ2 receptors was slower than that of α1β2γ2 for a wide range of GABA concentrations. Single-channel analysis did not reveal differences in the opening/closing kinetics of α1β2γ2 and α3β2γ2 channels but burst durations were longer in α3β2γ2 receptors. Simulation with a previously reported kinetic model was used to explore the differences in respective rate constants. Reproduction of major kinetic differences required a smaller desensitization rate as well as smaller binding and unbinding rates in α3β2γ2 compared with α1β2γ2 receptors. Our work describes the mechanisms underlying the kinetic differences between two major GABAA receptor subtypes and provides a framework to interpret data from native GABA receptors.

Extrasynaptic αβ subunit GABAA receptors on rat hippocampal pyramidal neurons

Extrasynaptic GABAA receptors that are tonically activated by ambient GABA are important for controlling neuronal excitability. In hippocampal pyramidal neurons, the subunit composition of these extrasynaptic receptors may include α5βγ and/or α4βδ subunits. Our present studies reveal that a component of the tonic current in the hippocampus is highly sensitive to inhibition by Zn2+. This component is probably not mediated by either α5βγ or α4βδ receptors, but might be explained by the presence of αβ isoforms. Using patch‐clamp recording from pyramidal neurons, a small tonic current measured in the absence of exogenous GABA exhibited both high and low sensitivity to Zn2+ inhibition (IC50 values, 1.89 and 223 μm, respectively). Using low nanomolar and micromolar GABA concentrations to replicate tonic currents, we identified two components that are mediated by benzodiazepine‐sensitive and ‐insensitive receptors. The latter indicated that extrasynaptic GABAA receptors exist that are devoid of γ2 subunits. To distinguish whether the benzodiazepine‐insensitive receptors were αβ or αβδ isoforms, we used single‐channel recording. Expressing recombinant α1β3γ2, α5β3γ2, α4β3δ and α1β3 receptors in human embryonic kidney (HEK) or mouse fibroblast (Ltk) cells, revealed similar openings with high main conductances (∼25–28 pS) for γ2 or δ subunit‐containing receptors whereas αβ receptors were characterized by a lower main conductance state (∼11 pS). Recording from pyramidal cell somata revealed a similar range of channel conductances, indicative of a mixture of GABAA receptors in the extrasynaptic membrane. The lowest conductance state (∼11 pS) was the most sensitive to Zn2+ inhibition in accord with the presence of αβ receptors. This receptor type is estimated to account for up to 10% of all extrasynaptic GABAA receptors on hippocampal pyramidal neurons.

An Interaction Involving an Arginine Residue in the Cytoplasmic Domain of the 5-HT3A Receptor Contributes to Receptor Desensitization Mechanism

A large cytoplasmic domain accounts for approximately one-third of the entire protein of one superfamily of ligand-gated membrane ion channels, which includes nicotinic acetylcholine (nACh), gamma-aminobutyric acid type A (GABA(A)), serotonin type 3 (5-HT3), and glycine receptors. Desensitization is one functional feature shared by these receptors. Because most molecular studies of receptor desensitization have focused on the agonist binding and channel pore domains, relatively little is known about the role of the large cytoplasmic domain (LCD) in this process. To address this issue, we sequentially deleted segments of the LCD of the 5-HT3A receptor and examined the function of the mutant receptors. Deletion of a small segment that contains three amino acid residues (425-427) significantly slowed the desensitization kinetics of the 5-HT3A receptor. Both deletion and point mutation of arginine 427 altered desensitization kinetics in a manner similar to that of the (425-427) deletion without significantly changing the apparent agonist affinity. The extent of receptor desensitization was positively correlated with the polarity of the amino acid residue at 427: the desensitization accelerates with increasing polarity. Whereas the R427L mutation produced the slowest desensitization, it did not significantly alter single channel conductance of 5-HT3A receptor. Thus, the arginine 427 residue in the LCD contributes to 5-HT3A receptor desensitization, possibly through forming an electrostatic interaction with its neighboring residues. Because the polarity of the amino acid residue at 427 is highly conserved, such a desensitization mechanism may occur in other members of the Cys-loop family of ligand-gated ion channels.

μ-δ Opioid Receptor Functional Interaction: Insight Using Receptor-G Protein Fusions

Fusion proteins between a receptor and a pertussis toxin-insensitive G(i)alpha subunit were used to gain insight into the molecular interactions that take place upon mu and delta opioid receptor heterodimerization. When mu opioid receptor-G(i1)alpha fusions were coexpressed with nonfused delta opioid receptors in human embryonic kidney 293 cells, or vice versa, receptor heterodimers were detected by coimmunoprecipitation. In pertussis toxin-treated cells, receptor coexpression decreased the amount of guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) incorporated in the fused G alpha protein after the addition of agonists specific for the receptor-G(i1)alpha fusion. In addition, activation of the G alpha protein occurred in heterodimers upon addition of an agonist specific for the nonfused receptor. It remained unaffected by an inverse agonist specific for the receptor-G(i1)alpha fusion. These data suggest that signaling through the receptor-G(i1)alpha fusion protein is impaired in heterodimers and support a mechanism in which activation of the G alpha subunit is promoted by a direct interaction with the nonfused receptor. Alternatively, receptor coexpression did not modify the ligand binding properties for the high-affinity state of the receptor-G(i1)alpha fusion nor the EC50 values for agonist-induced [35S]GTPgammaS incorporation in the G(i1)alpha subunit. In addition, no binding competition was observed between delta and mu ligands. Together, the data point to mu-delta opioid receptor heterodimers formed by contact interactions between monomers that retain their structural integrity.

Structural Determinants of Pharmacological Specificity Between D1and D2Dopamine Receptors

To test the hypothesis that pharmacological differentiation between D(1) and D(2) dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D(2) receptor to the corresponding aligned residues in the D(1) receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D(2) receptors and 11 mutant D(1) receptors for D(1)- and D(2)-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D(1) receptor mutant W99(3.28)F showed dramatically increased affinity for several D(2)-selective antagonists, particularly spiperone (225-fold), whereas the D(2) receptor mutant Y417(7.43)W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D(1)-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V(5.39) caused modest but consistent and reciprocal changes in affinity of the receptors for D(1) and D(2)-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D(1)/D(2) receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.

Activation of Adenylyl Cyclase by Endogenous Gs-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT7Receptor Expression

Human 5-hydroxytryptamine(7) (5-HT(7)) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G(i/o) and G(q/11) are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT(7) receptor expression was sufficient to limit signaling of endogenously expressed G(s)-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT(4(b)) and 5-HT(7(a,b,d)) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G(s)-coupled beta-adrenergic (betaAR) and prostanoid EP (EPR) receptors. betaAR- and EPR-stimulated AC activity was attenuated by 5-HT(7) receptor expression in both membrane preparations and intact HEK293 cells. betaAR- and EPR-stimulated AC activity was unaffected by expression of the G(s)-coupled 5-HT(4) receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) betaAR- and EPR-stimulated AC activity was not restored to control values when Galpha(s) was overexpressed; and 2) beta(1)AR and beta(2)AR activation of Galpha(s) was unaffected by the expression of 5-HT(7) receptors. In addition, overexpression of AC isoforms was unable to rescue betaAR- and EPR-stimulated AC activity. Therefore, 5-HT(7) receptors probably limit access and/or impede activation of AC by betaAR and EP receptors. Although the 5-HT(7) receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.

Interaction of Bivalent Ligand KDN21 with Heterodimeric δ-κ Opioid Receptors in Human Embryonic Kidney 293 Cells

KDN21 is a bivalent ligand that contains delta and kappa opioid antagonist pharmacophores linked through a 21-atom spacer. It has been reported that KDN21 bridges delta and kappa receptors that are organized as heterodimers. We have shown previously that when using [(3)H]diprenorphine as radioligand, KDN21 displayed greatly enhanced affinity in this series for coexpressed delta and kappa opioid receptors (CDK). The present study used in vitro expression systems to investigate interactions of members of the KDN series with delta-kappa heterodimers through competition binding using selective ligands and the mitogen-activated protein kinase (MAPK) assay. In this regard, the use of the selective radioligands [(3)H]naltrindole and [(3)H]norbinaltorphimine (nor-BNI) in competition binding studies revealed that KDN21 has much higher affinity than other KDN members for CDK and bound to CDK more selectively relative to mixed delta and kappa opioid receptors or singly expressed delta and kappa opioid receptors. Other experiments revealed that the binding of naltrindole to delta opioid receptors could increase the binding of nor-BNI to kappa opioid receptors and vice versa, suggesting reciprocal allosteric modulation of receptors in the heterodimer. Regarding the selectivity of KDN21 for phenotypic delta and kappa opioid receptors, we investigated the effect of KDN21 on the activation of MAPKs [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] by delta- or kappa-selective agonists. KDN21 inhibited the activation of ERK1/2 by [D-Pen(2),D-Pen(5)]-enkephalin (delta(1)) and bremazocine (kappa(2)) but had no effect on the activation by deltorphin II (delta(2)) and (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide (U69593, kappa(1)). 7-Benzylidenenaltrexone (delta(1)) and bremazocine (kappa(2)) significantly reduced the binding of KDN21 to CDK, whereas naltriben (delta(2)) and U69593 produced no such change. Taken together, these data support the idea that the organization of delta and kappa receptors as heterodimers gives rise to delta(1) and kappa(2) phenotypes.

Receptor Signaling and Endocytosis Are Differentially Regulated by Somatostatin Analogs

Upon hormone stimulation, the sst2 somatostatin receptor couples to adenylyl cyclase through G(i/o) proteins and undergoes rapid endocytosis via clathrin-coated pits. In this study, we determined the relationship between the ability of ligands to induce sst2 receptor internalization and inhibit adenylyl cyclase. Immunocytochemical studies demonstrated that peptide agonists [such as somatostatin-14, cortistatin-17, octreotide, vapreotide, KE108 (Tyr0-cyclo[d-diaminobutyric acid-Arg-Phe-Phe-d-Trp-Lys-Thr-Phe]), and SOM230 (cyclo[diaminoethylcarbamoyl-hydroxyproline-phenylglycine-d-Trp-Lys-(4-O-benzyl)-l-Tyr-Phe])] and nonpeptide agonists (such as L-779,976), stimulated the rapid endocytosis of sst2 receptors in human embryonic kidney 293 and CHO-K1 cells. In contrast, two antagonists did not induce receptor endocytosis by themselves and completely blocked agonist stimulation. Using a quantitative enzyme-linked immunosorbent assay to measure sst2 receptor sequestration, we found that peptide agonists varied by more than 100-fold in their potencies but exhibited the same efficacy as somatostatin14. In contrast, L-779,976 did not induce maximal receptor internalization. It is interesting that although betaarrestin-2 was recruited to cell surface sst2 receptors after stimulation with either somatostatin14 or L-779,976, the betaarrestin-receptor complex dissociated earlier in the endocytic pathway with the nonpeptide ligand. Although all agonists, including L-779,976, produced the same maximal inhibition of cyclic AMP, the potency ratio for inhibition of cyclic AMP and stimulation of receptor endocytosis varied by 15-fold. In general, native peptides showed similar potencies for cyclic AMP inhibition and receptor endocytosis, whereas short therapeutic analogs were substantially more potent at inhibiting cyclic AMP synthesis. These results demonstrate that the activity of somatostatin analogs to regulate receptor endocytosis and signaling are not tightly linked and provide compelling evidence for the induction of agonist specific states of the sst2 receptor.

Piglet saphenous vein contains multiple relaxatory prostanoid receptors: evidence for EP4, EP2, DP and IP receptor subtypes

Prostaglandin E(2) produced endothelium-independent relaxation of phenylephrine- and 5-HT-contracted piglet saphenous vein (PSV; pEC(50)=8.6+/-0.2; n=6). The prostanoid EP(4) receptor antagonist GW627368X (30-300 nM) produced parallel rightward displacement of PGE(2) concentration-effect (E/[A]) curves (pK(b)=9.2+/-0.2; slope=1). Higher concentrations of GW627368X did not produce further rightward shifts, revealing the presence of non-EP(4) prostanoid receptors. In all, 18 other prostanoid receptor agonists relaxed PSV in a concentration-related manner. Relative potencies of agonists most sensitive to 10 muM GW627368X (and therefore predominantly activating EP(4) receptors) correlated well with those at human recombinant EP(4) receptors in human embryonic kidney (HEK-293) cells (r(2)=0.74). In the presence of 10 microM GW627368X, the rank order of agonist relative potency matched that of the human recombinant EP(2) receptor in Chinese hamster ovary cells (r(2)=0.72). Iloprost, cicaprost and PGI(2) relaxed PSV maximally and were antagonised by 10 microM GW627368X, demonstrating that they were full EP(4) receptor agonists. Residual responses to these compounds in the presence of GW627368X suggested the presence of IP receptors.BW245C relaxed PSV maximally (pEC(50)=6.8+/-0.1). In the presence of 10 microM GW627368X, BW245C produced biphasic E/[A] curves (phase one pEC(50)=6.6; alpha=24%; phase two pEC(50)=5.1; alpha=112%). Phase two was antagonised by the DP receptor antagonist BW A868C (1 microM), demonstrating that BW245C is an agonist at DP and EP4 receptors. We conclude that PSV contains EP(4), EP(2), DP and IP receptors; IP receptor agonists are also porcine EP(4) receptor agonists.

Interactions of Postsynaptic Density-95 and the NMDA Receptor 2 Subunit Control Calpain-Mediated Cleavage of the NMDA Receptor

The calcium-dependent protease calpain cleaves the NMDA receptor 2 (NR2) subunit of the NMDA receptor both in vitro and in vivo and thus potentially modulates NMDA receptor function and turnover. We examined the ability of postsynaptic density-95 (PSD-95) protein to alter the calpain-mediated cleavage of NR2A and NR2B. Coexpression of PSD-95 with NMDA receptors in human embryonic kidney 293 cells blocked cleavage of NR2A and NR2B by NMDA receptor-activated calpain. NR2A cleavage by calpain occurred in the cell surface and intracellular fractions and required the presence of NR1 subunits. The blocking effect of PSD-95 did not result from decreased calpain activity, lowered intracellular calcium responses, or the blockade of internalization. Instead, this effect was eliminated by deletion of the C-terminal ESDV motif of NR2A or by overexpression of a palmitoylation-deficient PSD-95 mutant lacking the ability to cluster and to interact with NMDA receptors in situ, suggesting a role for association between the C terminus of NR2A and clustered PSD-95. Synapse-associated protein 102, a membrane-associated guanylate kinase interacting with NR2A but lacking palmitoylation motifs and the ability to cluster, did not protect NR2A from cleavage by calpain. Pharmacological inhibition of palmitoylation disrupted the interaction of PSD-95 with NMDA receptors in cortical neurons and allowed NR2A to be cleaved by calpain, whereas NR2A could not be cleaved in untreated neurons. These results indicate that PSD-95 clustering and direct association of NR2A and PSD-95 mediate the blocking effect of PSD-95 on calpain cleavage. PSD-95 could regulate the susceptibility of NMDA receptors to calpain-mediated cleavage during synaptic transmission and excitotoxicity.

An arginine involved in GABA binding and unbinding but not gating of the GABA(A) receptor.

GABA(A) receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose-response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function. Here, we focused on a residue (beta2-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of beta2-R207, we expressed alpha1beta2 and alpha1beta2-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because beta2-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

Arginine 222 in the Pre-transmembrane Domain 1 of 5-HT3A Receptors Links Agonist Binding to Channel Gating

Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3A receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3A receptors.

Agonist-dependent internalization of the human melanocortin-4 receptors in human embryonic kidney 293 cells.

A chimeric protein comprised of melanocortin-4 receptor (MC4R) and the green fluorescent protein (GFP) was created for studying receptor/ligand localization and trafficking. The ligand binding affinities and second messenger stimulation induced by MC4R-GFP closely resembled those of the wild-type receptor, suggesting functional integrity of the chimeric protein. As observed with a confocal microscope, in human embryonic kidney (HEK)-293 cells MC4R/GFP was distributed evenly along the cell membrane. Addition of [Nle4-d-Phe7]-alpha-melanocyte-stimulating hormone (NDP-MSH), a peptide MC4R agonist, induced receptor translocation into intracellular compartments in a time- and concentration-dependent manner. [Ac-Nle-c[Asp-His-d-Nal(2')-Arg-Trp-Lys]-NH2] (SHU9119), a potent MC4R antagonist, completely inhibited NDP-MSH-mediated internalization. MC4R-GFP internalization was unaffected by a protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), but was impaired by pretreatment with inhibitors of endocytosis through clathrin-coated pits, hypertonic sucrose, or concanavalin A. Time-dependent colocalization of MC4R-GFP with rhodamine-transferrin, an early endosome marker, and with LysoTraker, a lysosome marker, was observed after short-term (45 min) and prolonged (20 h) agonist exposure, respectively. Rhodamine-[AcNle-c[Asp-His-d-Phe-Arg-Trp-Lys]-NH2] (MTII), a fluorescent derivative of an MC4R agonist, was found to cointernalize with MC4R-GFP into intracellular vesicles. No significant receptor recycling or segregation from the ligand was observed 60 min after removal of the agonist. In contrast, an antagonist rhodamine-Ac-Cys-Glu-His-(d-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH2 (HS014) bound to and colocalized with MC4R-GFP on the cell surface and did not stimulate receptor internalization. In sum, these results suggest that MC4R is subject to agonist-dependent endocytosis via clathrin-coated pits. Prolonged agonist exposure directs MC4R into lysosomes, possibly for degradation. Receptor and ligand recycling is not efficient for MC4R in HEK-293 cells.

Identification of a testicular odorant receptor mediating human sperm chemotaxis.

Although it has been known for some time that olfactory receptors (ORs) reside in spermatozoa, the function of these ORs is unknown. Here, we identified, cloned, and functionally expressed a previously undescribed human testicular OR, hOR17-4. With the use of ratiofluorometric imaging, Ca2+ signals were induced by a small subset of applied chemical stimuli, establishing the molecular receptive fields for the recombinantly expressed receptor in human embryonic kidney (HEK) 293 cells and the native receptor in human spermatozoa. Bourgeonal was a powerful agonist for both recombinant and native receptor types, as well as a strong chemoattractant in subsequent behavioral bioassays. In contrast, undecanal was a potent OR antagonist to bourgeonal and related compounds. Taken together, these results indicate that hOR17-4 functions in human sperm chemotaxis and may be a critical component of the fertilization process.

Further characterization of the molecular interaction between PSD-95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating.

Coexpression of PSD-95(c-Myc) with NR1-1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3 H]MK801 binding similar to that previously described for l-glutamate. The inhibition constants (K (I) s) for the binding of l-glutamate and glycine to NR1-1a/NR2A determined by [3 H]CGP 39653 and [3 H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1-1a/NR2A receptors coexpressed +/- PSD-95(c-Myc). The increased EC(50) for l-glutamate enhancement of [3 H]MK801 binding was also found for NR1-2a/NR2A and NR1-4b/NRA receptors thus the altered EC(50) is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1-4b but not the NR1-1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1-4b and NR1-4b/NR2A expression was enhanced by PSD-95(c-Myc) but whole cell enzyme-linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD-95(c-Myc) has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C-terminal E(T/S)XV PSD-95 binding motifs.

Metabotropic glutamate receptor 1 internalization induced by muscarinic acetylcholine receptor activation: differential dependency of internalization of splice variants on nonvisual arrestins.

In this study, we characterized the glutamate- or second-messenger kinase-dependent internalization of the rat metabotropic glutamate receptor 1 (mGluR1) splice variants 1a, 1b, and 1c, and assessed the arrestin and dynamin dependence of these processes. To facilitate this we inserted a hemagglutinin epitope tag in the extracellular N-terminal domain of the splice variants. Quantification of glutamate-induced mGluR1 splice variant internalization provided by enzyme-linked immunosorbent assay and confirmed by immunofluorescent microscopy indicated that each splice variant underwent rapid internalization, which was strongly inhibited by coexpression of dominant-negative mutant (DNM) arrestin or dynamin. In addition glutamate-induced rapid translocation of arrestin-2-green fluorescent protein (GFP) or arrestin-3-GFP from cytosol to membrane was observed in cells expressing mGluR1 splice variants. Glutamate-induced internalization of mGluR1a and mGluR1c was partially blocked by a selective inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF 109203X), whereas mGluR1b internalization was not significantly affected by this inhibitor. Similarly, inositol phosphate production after glutamate-induced activation of mGluR1a and mGluR1c was increased after PKC inhibition, whereas glutamate-induced mGluR1b stimulation was unaffected. Activation by carbachol of endogenously expressed M(1) muscarinic receptors in human embryonic kidney 293 cells, induced the internalization of mGluR1 splice variants, which was partially blocked by pretreatment with inhibitors of either PKC or Ca(2+) calmodulin-dependent kinase II (CaMKII). Expression of DNM-arrestin with mGluR1a or 1c strongly inhibited carbachol-induced internalization. However, coexpression of DNM-arrestin with mGluR1b was less effective in reducing carbachol-induced receptor internalization. In addition, arrestin-2-GFP or arrestin-3-GFP underwent significant carbachol-induced translocation from cytosol to membrane in cells coexpressing mGluR1a or 1c but not in cells coexpressing mGluR1b. This study demonstrates that the internalization of mGluR1 splice variants is subject to PKC and CaMKII regulation. In addition, regulation by these kinases confers differential arrestin dependence.

Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human alpha(1)beta glycine receptors.

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR beta subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the beta subunit was examined using site-directed mutagenesis and expression of recombinant (alpha(1)beta(Y413F)) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents (I(Gly)) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I(Gly) in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC(50) = 143 microM; cSrc EC(50) = 74 microM). Exogenous cSrc, applied intracellularly, upregulated heteromeric alpha(1)beta receptors but not homomeric alpha(1) receptors. Substitution mutation of the tyrosine to phenylalanine at position beta-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type alpha(1)beta but not alpha(1)beta(Y413F) receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the beta subunit.

The Inhibitory γ Subunit of the Type 6 Retinal Cyclic Guanosine Monophosphate Phosphodiesterase Is a Novel Intermediate Regulating p42/p44 Mitogen-activated Protein Kinase Signaling in Human Embryonic Kidney 293 Cells

The inhibitory gamma subunits of the retinal rod and cone photoreceptor type 6 retinal cyclic guanosine monophosphate phosphodiesterase (PDEgamma) are expressed in non-retinal tissues. Here, we show that PDEgamma interacts with the G-protein-coupled receptor kinase 2 signaling system to regulate the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. This is based upon several lines of evidence. First, the transfection of cells with an antisense rod PDEgamma plasmid construct, which reduced endogenous rod PDEgamma expression, ablated the epidermal growth factor- and thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase. Second, the transfection of cells with recombinant rod or cone PDEgamma and/or G-protein-coupled receptor kinase 2 increased the stimulation of p42/p44 mitogen-activated protein kinase by epidermal growth factor or thrombin. In contrast, a G-protein-coupled receptor kinase 2 phosphorylation-resistant rod PDEgamma mutant failed to increase the epidermal growth factor- or thrombin-dependent stimulation of p42/p44 mitogen-activated protein kinase and, in fact, functioned as a dominant negative. Thrombin also stimulated the association of endogenous rod PDEgamma with dynamin II, which was increased in cells transfected with rod PDEgamma or G-protein-coupled receptor kinase 2. Dynamin II plays a critical role in regulating endocytosis of receptor signal complexes required for activation of p42/p44 mitogen-activated protein kinase. Therefore, PDEgamma may have an important role in promoting endocytosis of receptor signal complexes leading to the activation of p42/p44 mitogen-activated protein kinase. We conclude that PDEgamma is an entirely novel intermediate regulating mitogenic signaling from both receptor tyrosine kinase and G-protein-coupled receptors in human embryonic kidney 293 cells.

NF449: a subnanomolar potency antagonist at recombinant rat P2X1 receptors.

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC50<3.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 >> P2X3 > P2Y1 > P2Y2 > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.