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W Sadée

Publications and source records attributed to W Sadée.

At least 55 records · Page 3Linked to original sources
PubMed1997

A constitutively internalizing and recycling mutant of the mu-opioid receptor.

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in mu-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (354ThrSerSerThrIleGluGlnGlnAsn362) unique to the C-terminus of the mu-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these mu-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wild-type and mutant receptors. Both the wild-type mu-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress mu-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.

Analgesics↗
PubMed1996

Basal phosphorylation of mu opioid receptor is agonist modulated and Ca2+-dependent.

The mu opioid receptor was shown to be phosphorylated at a basal rate in the absence of agonist, measured in permeabilized HEK293 cells transfected with an epitope tagged mu receptor (EE-mu) [Arden, J., Segredo, V., Wang, Z., Lameh, J. and Sadee, W. (1995) J. Neurochem. 65, 1636-1645]. In the present study, basal phosphorylation was found to be Ca2+ dependent; however, several inhibitors of protein kinase C and Ca2+-calmodulin dependent kinases failed to affect basal mu receptor phosphorylation. Thus, the basal mu receptor phosphorylating activity differed from the main kinases involved in receptor regulation. The general kinase inhibitor H7 (100 microM) suppressed basal mu receptor phosphorylation. Pretreatment with the agonist morphine, followed by drug removal, resulted in a sustained increase of basal mu receptor phosphorylation. The gradual agonist dependent modulation of basal mu receptor phosphorylation suggests a novel regulatory mechanism which may play a role in narcotic tolerance and dependence.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice.

N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to block the development of antinociceptive tolerance to morphine. Assessment of the effects of NMDA antagonists on development of antinociceptive tolerance to selective opioid mu (mu) and delta (delta) agonists, however, has not been reported. In these experiments, selective mu and delta receptor agonists, and morphine, were repeatedly administered to mice either supraspinally (i.c.v.) or systemically (s.c.), alone or after pretreatment with systemic NMDA antagonists. Antinociception was evaluated using a warm-water tail-flick test. Repeated i.c.v. injections of mu agonists including morphine, fentanyl, [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) and Tyr-Pro-NMePhe-D-Pro-NH2 (PL017) or [D-Ala2, Glu4]deltorphin, a delta agonist, or s.c. injections of morphine or fentanyl, produced antinociceptive tolerance as shown by a significant rightward displacement of the agonist dose-response curves compared to controls. Single injections or repeated administration of MK801 (a non-competitive NMDA antagonist) or LY235959 (a competitive NMDA antagonist) at the doses employed in this study did not produce behavioral toxicity, antinociception or alter the acute antinociceptive effects of the tested opioid agonists. Consistent with previous reports, pretreatment with MK801 or LY235959 (30 min prior to agonist administration throughout the tolerance regimen) prevented the development of antinociceptive tolerance to i.c.v. or s.c. morphine. Neither NMDA antagonist, however, affected the development of antinociceptive tolerance to i.c.v. fentanyl, DAMGO, or [D-Ala2, Glu4]deltorphin. Additionally, MK801 pretreatment did not affect the development of antinociceptive tolerance to i.c.v. PL017 or to s.c. fentanyl. Further, MK801 pretreatment also did not affect the development of tolerance to the antinociception resulting from a cold-water swim-stress episode, previously shown to be a delta-opioid mediated effect. These data lead to the suggestion that the mechanisms of tolerance to receptor selective mu and delta opioids may be regulated differently from those associated with morphine. Additionally, these findings emphasize that conclusions reached with studies employing morphine cannot always be extended to 'opiates' in general.

Analgesics, Opioid↗
PubMed1996

Structure-based search for peptide ligands that cross-react with melanocortin receptors.

PURPOSE: To define sequence motifs that can be used to identify peptide ligands of the melanocortin receptor (MCR). METHODS: Screening of combinatorial libraries has led to identification of D-Trp-Nle-NH2 (Nle, norleucine) and D-Trp-Arg-NH2 as the smallest structures known to antagonize the amphibian MCR (1). As the basis of a search paradigm, peptide-ligands containing these or similar motifs within their larger primary structure were examined for ability to antagonize amphibian and recombinant human MCRs. Compounds examined include analogs of substance P, leutinizing-hormone releasing-hormone, endothelin, neurotensin, and opioid-somatostatin. RESULTS: Of seven compounds tested containing the predetermined search motif D-Trp-AAx (where AAx is Arg, Leu, Nle, or Ile), six were found to have previously unrecognized antagonist activity at the amphibian MCR (Kd 30 to 5000 nM). In contrast, of 14 similar control peptides lacking the D-Trp-AAx search motif, only somatostatin displayed measurable antagonist potency. The anticancer peptide, [Arg8, D-Trp7.9, N-methyl-Phe8]-substance P, was the most potent of the motif-containing peptides with a Kd of 31 nM. The mu-opioid antagonist D-Phe-cyclic[Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH2 (CTAP) also blocked the amphibian MCR (Kd 1 microM), but the related mu-antagonist CTOP, different only by only by substitution of Arg with ornithine within the search motif, was found to agonize the amphibian MCR (EC50 67 nM). CTAP and the anticancer peptide were also tested on human MCRs (hMCRs); while CTAP competed with alpha-MSH at the hMC1 receptor, the anticancer peptide had no effect or was slightly stimulatory. CONCLUSIONS: We have identified dipeptide motifs that help distinguish antagonist ligands of the amphibian MCR from ligands known to interact with other G-protein coupled receptors. This approach might be generally applicable if motifs can identified for other receptors and their subtypes. In studies employing CTAP and CTOP, analogs previously considered highly selective for the mu-opioid receptor, cross-reaction with MCRs must be considered.

Analgesics↗
PubMed1996

Human dipeptide transporter, hPEPT1, stably transfected into Chinese hamster ovary cells.

PURPOSE: A cDNA encoding the H(+)-coupled peptide transporter, hPEPT1, has previously been cloned from human ileum (8). The objective of this study was to establish a stably transfected cell line expressing hPEPT1 in mammalian cell culture. METHODS: The hPEPT1 cDNA was subcloned into an expression vector carrying the CMV promoter and a neomycin resistance gene. This vector, pCDNA3-PEPT1, was transiently transfected into several cell lines to identify those capable of expressing PEPT1 transport function. CHO cells were selected and stably transfected with PEPT1 (CHO-PEPT1). Dipeptide transport activity was measured with 3H-Gly-Sar, in the presence and absence of inhibitors. RESULTS: The clonal cell line, CHO-PEPT1, displayed high transport activity. Dipeptide transport was sensitive to pH and specific for dipeptides and other small peptides. Peptidomimetic antibiotics, such as cephalexin, were competitors for peptide transport. CONCLUSIONS: The stably transfected cell line, CHO-PEPT1 exhibits enhanced transport over that of cell lines with native expression of PEPT1, and therefore, represents a useful tool for rapid screening of drugs that utilize the peptide transporter in the human intestine for absorption.

Ampicillin↗
PubMed1996

Effects of naloxone and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 and the protein kinase inhibitors H7 and H8 on acute morphine dependence and antinociceptive tolerance in mice.

Previous studies measuring opioid inhibition of cyclic adenosine monophosphate in SH-SY5Y cells supported the hypothesis that continuous agonist stimulation causes a gradual conversion of the mu opioid receptor to a sensitized or constitutively active state termed mu*. Conversion to mu* was prevented by the kinase inhibitor H7, but not its close analog H8. Naloxone was proposed to act as a negative antagonist (inverse agonist) blocking mu* activity, whereas D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) appeared to act as a neutral antagonist having no effect on mu* activity. Initial in vivo results indicated that mu* activity may play a role in narcotic tolerance and dependence (Wang et al., Life Sci. 54: PL339-PL350 1994). Our study explores the pharmacology of H7 and H8, naloxone and CTAP in mice after induction of acute tolerance and dependence induced by a single s.c. dose of morphine (100 mg/kg). Physical dependence was defined by withdrawal jumping induced by i.p. naloxone injections 4 hr after the morphine dose, the time of maximal physical dependence. Neither H7 nor H8 (50 nmol or less) induced jumping, affected morphine antinociception or produced significant behavioral effects, when injected by the intracerebroventricular (i.c.v.) or intrathecal (i.th.) routes. When given 30 min before the naloxone challenge, H7, but not H8, significantly reduced naloxone jumping by i.c.v. injection. Administration of naloxone into the central nervous system, rather than by i.p. administration, required coinjection by both i.c.v. and i.th. routes to elicit full withdrawal jumping (30 nmol at each site). In contrast, the putative neutral antagonist CTAP caused little withdrawal jumping when coinjected i.c.v. and i.th., as expected if modulation of mu* activity played a role in dependence. However, CTAP was capable of partially reversing naloxone (i.p.) induced jumping when given either i.c.v. or i.th., indicating that CTAP competes with naloxone at mu*. Moreover, these results demonstrate that both spinal and supraspinal sites are required for full opioid withdrawal jumping in mice. Antinociceptive tolerance was also evaluated by determining the response to morphine in the 55 degrees C warm-water tail-flick test. Morphine pretreatment (100 mg/kg, s.c., -5 hr) produced antinociceptive tolerance as shown by a 2.7-fold increase in the calculated morphine A50 value. Tolerance was reversed by H7, but not H8, treatment (50 nmol, i.c.v., -30 min). These results are consistent with the hypothesis that a sensitized or constitutively active mu* state plays a role in narcotic tolerance and dependence.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Activating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine Hm1 receptors.

The N- and C-terminal junctions of the third intracellular loop (i3) of G protein-coupled receptors play a role in the coupling process. We had previously constructed two triple point alanine mutants of the i3 junction of the muscarinic Hm1 receptor, W209A/I211A/Y212A and E360A/K362A/T366A, which are defective in mediating carbachol stimulation of phosphatidylinositol (PI) turnover (Moro, O., Lameh, J., Högger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276). Each of the corresponding six single point mutations were constructed to determine residues crucial to receptor coupling. Mutants W209A and T366A were similar to or only slightly less effective than wild type Hm1 in stimulating PI turnover. In the N-terminal junction, I211A and Y212A were defective in coupling, and I211A was even more defective than the corresponding triple mutant. Therefore, the triple mutation compensated at least partially for the effect of these two single point mutations. In the C-terminal i3 loop junction, mutant K362A was again more strongly defective than the corresponding triple mutant. In contrast, mutation E360A was found to be activating, leading to elevated PI turnover in the absence of agonist and sensitization toward carbachol activation. Activating mutations in the C-terminal i3 loop junction have been reported previously for the adrenergic receptors, but E360A represents the first muscarinic receptor with substantial basal activity. The effects of the single point mutations observed in this study were not readily predictable from similar mutations from closely related G protein-coupled receptors despite sequence conservation in the i3 loop junctions. Our results caution against defining precise coupling domains in these regions by mutagenesis results.

Alanine↗
PubMed1995

Agonist induced constitutive receptor activation as a novel regulatory mechanism. Mu receptor regulation.

We propose the hypothesis that certain G protein coupled receptors can become constitutively activated during agonist stimulation so that the receptor remains active even after the agonist is removed. This new paradigm of receptor regulation may account for some long term effects of neurotransmitters and hormones. We have tested the hypothesis that constitutive mu receptor activation represents a crucial step driving narcotic tolerance and dependence. Our results indeed support the conversion of mu to a constitutively active state, mu*, observed in neuroblastoma SK-N-SH and SH-SY5Y tissue culture, in U293 cells transfected with the mu receptor gene, and in vivo. Constitutive mu activation may result from receptor phosphorylation to yield mu*, and further, in vivo studies indicate that formation of mu* could account for narcotic tolerance and dependence.

Animals↗
PubMed1995

Biology of membrane transport proteins.

Membrane transporter proteins are encoded by numerous genes that can be classified into several superfamilies, on the basis of sequence identity and biological function. Prominent examples include facilitative transporters, the secondary active symporters and antiporters driven by ion gradients, and active ABC (ATP binding cassette) transporters involved in multiple-drug resistance and targeting of antigenic peptides to MHC Class I molecules. Transported substrates range from nutrients and ions to a broad variety of drugs, peptides and proteins. Deleterious mutations of transporter genes may lead to genetic diseases or loss of cell viability. Transporter structure, function and regulation, genetic factors, and pharmaceutical implications are summarized in this review.

ATP-Binding Cassette Transporters↗
PubMed1995

Phosphorylation and agonist-specific intracellular trafficking of an epitope-tagged mu-opioid receptor expressed in HEK 293 cells.

We expressed the cloned mu-opioid receptor (muR) in high abundance (5.5 x 10(6) sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged receptor (EE-muR) was similar to the untagged mu R ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the mu-specific peptide agonist [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-muR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-muR-containing membranes were solubilized in detergent [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate] and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at approximately 70 kDa with weaker bands at approximately 65 kDa. EE-muR was labeled with [gamma-32P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65-70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced approximately 1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the muR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.

Animals↗
PubMed1994

Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

Animals↗
PubMed1994

Overlapping multi-site domains of the muscarinic cholinergic Hm1 receptor involved in signal transduction and sequestration.

Alanine mutagenesis scanning of the intracellular portion of the human muscarinic cholinergic Hm1 receptor was performed to identify domains mediating agonist induced receptor sequestration. Using these multiple alanine point mutants of Hm1, we had previously identified several receptor domains in the intracellular loops i1-3 that play a role in coupling to phosphatidyl inositol turnover, most notably, a lipophilic residue, Leu-131, in the conserved i2 loop domain DRYXXVXXPL (Moro, O., Lameh, J., Hogger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 6862-6865). We now demonstrate that alanine substitutions in three of these domains, i.e. middle of the i2 loop and both junctions of the i3 loop, also result in defective sequestration (loss of surface receptor sites accessible to a polar tracer) in transfected human kidney U293 cells. The i2 loop was studied further by single point mutations. The strongest impairment of sequestration occurred with mutant L131A which was also highly defective in phosphatidyl inositol (PI) coupling. Substitution of Leu-131 with several distinct amino acids indicated that a bulky lipophilic residue is required for sequestration in this position, as shown for coupling to PI turnover. Further, the double point mutation, V127A/L131A, almost completely suppressed both sequestration and coupling of Hm1. In the beta 2 adrenoceptor, alanine substitution of the i2 residue Phe-139, equivalent to Leu-131 in Hm1, also resulted in impaired coupling to adenylyl cyclase and sequestration, indicating a general role for this conserved i2 loop residue in both processes. The combined results show that the multi-site domain involved in signal transduction of Hm1 is similar to and overlaps with that involved in sequestration. However, three Hm1 mutants that were moderately deficient in stimulating PI turnover displayed normal sequestration, suggesting distinct mechanisms. We propose that cellular mediators of receptor sequestration are structurally similar or identical to the heterotrimeric G proteins.

Alanine↗
PubMed1994

Constitutive mu opioid receptor activation as a regulatory mechanism underlying narcotic tolerance and dependence.

Chronic administration of narcotic mu opioid agonists results in tolerance and dependence. We propose that agonist stimulation causes a gradual conversion of mu receptors to a constitutively active state (mu*) as a key step in tolerance and physical dependence. We provide evidence in support of the existence of mu* in human neuroblastoma cells, SH-SY5Y, and mu* upregulation during morphine treatment. Naloxone blocked mu* activity, acting as an antagonist with negative intrinsic activity which accounts for its high potency in eliciting withdrawal. In contrast, the mu selective antagonist CTAP did not affect mu* activity but inhibited naloxone's effect. The protein kinase inhibitor H7 was found to suppress mu* formation, suggesting that mu* is phosphorylated. In a model of acute morphine tolerance/dependence in mice, H7 prevented naloxone induced withdrawal jumping and reversed morphine (antinociceptive) tolerance. CTAP caused only mild withdrawal and attenuated naloxone induced withdrawal, as predicted for an antagonist without negative activity. These results support a role for constitutive mu receptor activation in narcotic tolerance and dependence, affording potential separation of acute and chronic narcotic effects.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling.

Signal transduction of the heptahelical G protein-coupled receptors (GPCRs) involves multiple receptor domains, but a universal consensus domain for coupling has not yet been defined. Alanine mutagenesis scanning was performed on the intracellular loops and the COOH tail of the human muscarinic cholinergic receptor (Hm1) to identify coupling domains. Stimulation of phosphatidylinositol (PI) turnover was determined after transfection of the alanine mutants into U293 human embryonic kidney cells. Alanine substitutions in four regions (loops i1, i2, and NH2 and COOH junctions of i3) impaired coupling efficiency by approximately 50% or more, but the strongest reduction (> 80%) resulted from alanine replacement of a single amino acid, leucine 131. This residue is located in the middle of the second intracellular loop (i2), within the highly conserved GPCR motif (DRYXXV(I)XXPL). The position equivalent to Leu-131 in Hm1 contains a bulky hydrophobic amino acid (L, I, V, M, or F) in nearly all cloned GPCRs. Substitution of Leu-131 with polar amino acids (aspartate and asparagine) also resulted in strongly defective coupling, whereas phenylalanine (found in the equivalent position in the beta 2 adrenoceptor) can replace leucine without losing PI coupling ability of Hm1. Alanine substitution of the corresponding amino acid in the Hm3 receptor (L174A) also inhibited agonist-stimulated PI turnover, while replacing Phe-139 with alanine in the beta 2 adrenoceptor suppressed stimulation of adenylyl cyclase. We propose that a bulky hydrophobic amino acid in the middle of the i2 loop serves as a general site relevant to G protein coupling, whereas coupling selectivity is governed by other receptor domains.

Amino Acid Sequence↗
PubMed1993

Serine- and threonine-rich domain regulates internalization of muscarinic cholinergic receptors.

Upon agonist exposure, most membrane receptors internalize into the cell as part of an adaptation process. Receptor domains that mediate internalization have been defined for several receptor classes, e.g. growth factor and transport receptors, but not yet for any of the numerous members of the family of G protein-coupled receptors (GPCRs), having seven putative transmembrane helices. With the use of deletion mutations, we previously showed that a small region in the middle of the third intracellular loop (i3) appears to be required for the agonist-induced internalization of the Hm1 muscarinic cholinergic receptor (Lameh, J., Philip, M., Sharma, Y. K., Moro, O., Ramachandran, J., and Sadée, W. (1992) J. Biol. Chem. 267, 13406-13412). Using point mutations, we now demonstrate that domain 286-292 (ESLTSSE) is required, with TSS playing a crucial role. Although the i3 loops of GPCRs share minimal overall sequence identity, even among closely related subtypes, small domains containing multiple S/T residues are also present in several other GPCRs, e.g. peptide, catechol, and all the muscarinic cholinergic receptor subtypes. S/T-->A mutations in analogous i3 domains also prevented receptor internalization in the case of Hm3 and attenuated internalization of Hm2. We propose that an S/T-rich domain in the i3 loop, possibly via phosphorylation reactions, regulates one pathway of GPCR internalization.

Amino Acid Sequence↗
PubMed1992

Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor.

We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling. Point mutations were constructed to test the role of charged amino acids in these junctions. A triple point mutation of Hm1 (E214 A/ E216K/ E221 K), which mimics the charge distribution in Hm2 (negatively coupled to cAMP) over the first 14 amino acids of i3, and a double point mutation in the N terminal junction, K359A/K361A, both failed to affect carbachol stimulated PI turnover. Therefore, charge distribution in the loop junctions appears to play a minor role in G protein coupling of Hm1 in HEK 293 cells.

Amino Acid Sequence↗
PubMed1992

Interaction among mu-opioid receptors and alpha 2-adrenoceptors on SH-SY5Y human neuroblastoma cells.

The clonal human neuroblastoma cell line SK-N-SH-SY5Y was previously shown to express mu-opioid and alpha 2-adrenoceptors which are both negatively coupled to adenylyl cyclase. Because of the potential use of alpha 2-agonists in the treatment of narcotic dependence, we tested the interactions among he alpha 2-agonists, clonidine and norepinephrine, and morphine on AC in SH-SY5Y cells. Pretreatment with retinoic acid resulting in partial neuronal differentiation greatly enhanced the cells' sensitivity towards adenylyl cyclase stimulation by prostaglandin E1, and its inhibition by morphine and alpha 2-agonists. Norepinephrine (EC50 = 69 nM) maximally inhibited prostaglandin E1-stimulated cAMP accumulation (by approximately 83%), and the alpha 2-agonist yohimbine reversed these effects. Clonidine (EC50 = 32 nM) was a partial agonist, with 50 to 60% maximal inhibition. The combined effects of morphine (maximum approximately 70% inhibition) and norepinephrine exceeded the effect of either agent alone, yielding more than 90% inhibition of prostaglandin E1-stimulated cAMP accumulation. As previously reported for morphine, only a partial tolerance was observed for adenylyl cyclase inhibition by norepinephrine. Further, no cross-tolerance was observed between clonidine and morphine. The combined results indicate that mu-opioid receptors and an alpha 2-adrenoceptor subtype are colocalized on the same cells in SH-SY5Y culture, which hence serves as a model to study opioid-alpha 2-adrenergic interactions.

Adenylyl Cyclase Inhibitors↗