Isolation, sequence, and functional expression of the mouse M1 muscarinic acetylcholine receptor gene

A genomic clone encoding the gene for the mouse M1 muscarinic acetylcholine receptor has been isolated, placed under the control of the zinc-inducible mouse metallothionein promoter, and transfected into mouse Y1 adrenal cells. The receptor concentration was about 300 fmol/mg membrane protein in the absence of zinc and could be increased to 4000 fmol/mg membrane protein in the presence of increasing concentrations of zinc. The receptor expressed in zinc-induced cells exhibits the high affinity binding for quinuclidinyl benzilate, atropine, and pirenzepine expected of the M1 muscarinic receptor. The M1 receptor when expressed in Y1 or L cells is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism, but does not inhibit forskolin stimulation of cAMP accumulation. In contrast, a cloned M2 muscarinic receptor when expressed in Y1 cells is able to inhibit forskolin stimulation of cAMP accumulation, but is unable to stimulate phosphatidylinositol metabolism. The stimulation of phosphatidylinositol metabolism mediated by the M1 receptor was not altered by prior treatment of Y1 cells with concentrations of islet-activating protein sufficient to eliminate M2 receptor-mediated inhibition of adenylate cyclase. The cloned M1 receptor gene thus exhibits both the pharmacological and physiological properties expected of the M1 muscarinic acetylcholine receptor. In addition, these results indicate that different subtypes of the muscarinic receptor are coupled to different physiological responses.     

NMDA Receptor Subtype Selectivity: Eliprodil, Polyamine Spider Toxins, Dextromethorphan, and Desipramine Selectively Block NMDA-Evoked Striatal Acetylcholine but Not Spermidine Release

Abstract: NMDA receptor stimulation concomitantly increases the release of [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The NMDA-induced release of both acetylcholine and spermidine was blocked with equal potency by the NMDA channel blocker phencyclidine (0.1–10 µ M ). However, certain other channel blockers, including dextromethorphan (1–100 µ M ), which antagonized NMDA-evoked acetylcholine release without affecting NMDA-evoked spermidine release, and dextrorphan (1–100 µ M ) and memantine (1–100 µ M ), which block NMDA-evoked acetylcholine release more potently than NMDA-evoked spermidine release, showed greater selectivity of action. As previously shown for ifenprodil, eliprodil (SL82.0715; 1–100 µ M ) blocked NMDA-evoked acetylcholine but not spermidine release. This selectivity is also observed for other agents interacting with the polyamine site(s) on the NMDA receptor, including arcaine (1–1,000 µ M ), philanthotoxin₃₄₃, and argiotoxin₆₃₆ (10 µ M ) and was also noted for desipramine (1–100 µ M ). The NMDA-induced release of acetylcholine and spermidine is likely to be mediated by different native NMDA receptor subtypes, and several NMDA antagonists may be candidates for a selective action at a particular NMDA receptor subtype.     

Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice

Acetylcholine regulates perfusion of numerous organs via changes in local blood flow involving muscarinic receptor-induced release of vasorelaxing agents from the endothelium. The purpose of the present study was to determine the role of M?, M?, and M? muscarinic acetylcholine receptors in vasodilation of small arteries using gene-targeted mice deficient in either of the three receptor subtypes (M1R(-/-), M3R(-/-), or M5R(-/-) mice, respectively). Muscarinic receptor gene expression was determined in murine cutaneous, skeletal muscle, and renal interlobar arteries using real-time PCR. Moreover, respective arteries from M1R(-/-), M3R(-/-), M5R(-/-), and wild-type mice were isolated, cannulated with micropipettes, and pressurized. Luminal diameter was measured using video microscopy. mRNA for all five muscarinic receptor subtypes was detected in all three vascular preparations from wild-type mice. However, M(3) receptor mRNA was found to be most abundant. Acetylcholine produced dose-dependent dilation in all three vascular preparations from M1R(-/-), M5R(-/-), and wild-type mice. In contrast, cholinergic dilation was virtually abolished in arteries from M3R(-/-) mice. Deletion of either M?, M?, or M? receptor genes did not affect responses to nonmuscarinic vasodilators, such as substance P and nitroprusside. These findings provide the first direct evidence that M? receptors mediate cholinergic vasodilation in cutaneous, skeletal muscle, and renal interlobar arteries. In contrast, neither M? nor M? receptors appear to be involved in cholinergic responses of the three vascular preparations tested.     

Reduced macrophage recruitment,proliferation, and activation in colony-stimulating factor-1-deficient mice results in decreased tubular apoptosis during renal inflammation

Kidney tubular epithelial cell (TEC) death may be dependent on the number and activation state of macrophages (M phi) during inflammation. Our prior studies indicate that activated M phi release soluble mediators that incite TEC death, and reducing intrarenal M phi during kidney disease diminishes TEC apoptosis. CSF-1 is required for M phi proliferation and survival. We hypothesized that in the absence of CSF-1, M phi-mediated TEC apoptosis would be prevented during renal inflammation. To test this hypothesis, we evaluated renal inflammation during unilateral ureter obstruction in CSF-1-deficient (Csf1(op)/Csf1(op)) mice. We detected fewer M phi and T cells and less apoptotic TEC in the obstructed kidneys of Csf1(op)/Csf1(op) mice compared with wild-type (WT) mice. The decrease in intrarenal M phi resulted from diminished recruitment and proliferation, not enhanced apoptosis. CSF-1 enhanced M phi activation. There were far fewer activated (CD69, CD23, Ia, surface expression) M phi in obstructed CSF-1-deficient compared with WT obstructed kidneys. Similarly, bone marrow M phi preincubated with anti-CSF-1 receptor Ab or anti-CSF-1 neutralizing Ab were resistant to LPS- and IFN-gamma-induced activation. We detected fewer apoptotic-inducing molecules (reactive oxygen species, TNF-alpha, inducible NO synthase) in 1) M phi propagated from obstructed Csf1(op)/Csf1(op) compared with WT kidneys, and 2) WT bone marrow M phi blocked with anti-CSF-1 receptor or anti-CSF-1 Ab compared with the isotype control. Furthermore, blocking CSF-1 or the CSF-1 receptor induced less TEC apoptosis than the isotype control. We suggest that during renal inflammation, CSF-1 mediates M phi recruitment, proliferation, activation, and, in turn, TEC apoptosis.     

Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT1A angiotensin receptor

An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.     

No change in cortical muscarinic M2, M3 receptors or [35S]GTPgammaS binding in schizophrenia

Muscarinic M1, but not M4, receptors have been shown to be decreased in Brodmann's area (BA) 9 obtained postmortem from subjects with schizophrenia. This study extends that data by measuring levels of muscarinic M2 and M3 receptor protein and mRNAs in BA 9 and BA 40 from the same cohorts of subjects used in the study of M1 and M4 receptors. In addition, the ability of carbachol to stimulate muscarinic receptors that signal through the Gi/o G-proteins was measured in BA 9 from the same cohorts of subjects. There were no changes in levels of muscarinic M2 or M3 protein or M3 mRNA with diagnosis in either CNS region. M2 receptor mRNA could not be detected in BA 9 or BA 40. Finally, carbachol-stimulated GTPgammaS binding did not differ between the diagnostic cohorts in BA 9 (p = 0.64). These data add considerable weight to the argument that the muscarinic M1 receptor is the muscarinic receptor predominantly affected in BA 9 by the pathology of schizophrenia. Given the widespread changes in muscarinic receptors identified in the CNS of subjects of schizophrenia using functional neuroimaging it remains possible that receptors other than the M1 receptor may be altered in different CNS regions.     

Acidic amino acids flanking phosphorylation sites in the M2 muscarinic receptor regulate receptor phosphorylation, internalization, and interaction with arrestins

The studies reported here address the molecular events underlying the interactions of arrestins with the M(2) muscarinic acetylcholine receptor (mAChR). In particular, we focused on the role of receptor phosphorylation in this process. Agonist-dependent phosphorylation of the M(2) mAChR can occur at clusters of serines and threonines at positions 286-290 (site P1) or 307-311 (site P2) in the third intracellular loop (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). Phosphorylation at either P1 or P2 can support agonist-dependent internalization. However, phosphorylation at P2 is required for receptor interaction with arrestins (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-26389). The present study investigated the role of acidic amino acids between P1 and P2 in regulating receptor phosphorylation, internalization, and receptor/arrestin interactions. Mutation of the acidic amino acids at positions 298-300 (site A1) and/or 304-305 (site A2) to alanines had significant effects on agonist-dependent phosphorylation. P2 was identified as the preferred site of agonist-dependent phosphorylation, and full phosphorylation at P2 required the acidic amino acids at A1 or their neutral counterparts. In contrast, phosphorylation at site P1 was dependent on site A2. In addition, sites A1 and A2 significantly affected the ability of the wild type and P1 and P2 mutant receptors to internalization and to interact with arrestin2. Substitution of asparagine and glutamine for the aspartates and glutamates at sites A1 or A2 did not influence receptor phosphorylation but did influence arrestin interaction with the receptor. We propose that the amino acids at sites A1 and A2 play important roles in agonist-dependent phosphorylation at sites P2 and P1, respectively, and also play an important role in arrestin interactions with the M(2) mAChR.     

M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M(3) receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M(3)-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M(3)-receptor knockout mice, suggesting that contraction was predominantly due to M(3)-receptor activation. However, approximately 50-60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M(3)-receptor knockout mice, indicating that contraction in these tissues was also due to M(2)-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M(3)-receptor knockout mice by M(1)-receptor activation. Thus M(3)-receptor knockout mice provided unambiguous evidence that M(3) receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M(2) receptors in mouse stomach fundus and trachea.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

PD 102807, a novel muscarinic M4 receptor antagonist, discriminates between striatal and cortical muscarinic receptors coupled to cyclic AMP

In membranes of Chinese hamster ovary cells expressing the cloned human M1-M4 muscarinic receptor subtypes, PD 102807, a novel M4 selective antagonist, was found to counteract the M4 receptor-induced stimulation of [35S]-GTPgammaS binding to membrane G proteins with a pK(B) of 7.40, a value which was 63-, 33- and 10-fold higher than those displayed at M1 (pK(B) = 5.60), M2 (pK(B) = 5.88) and M3 (pK(B) = 6.39) receptor subtypes, respectively. In rat striatal membranes, PD 102807 antagonized the muscarinic inhibition of dopamine (DA) D1 receptor-stimulated adenylyl cyclase with a pK(B) value of 7.36. In contrast, in membranes of rat frontal cortex, PD 102807 displayed lower potencies in antagonizing either the muscarinic facilitation of corticotropin releasing hormone (CRH)-stimulated adenylyl cyclase (pK(B) = 5.79) or inhibition of Ca2+/calmodulin (Ca2+/CaM)-stimulated enzyme activity (pK(B) = 5.95). In each response investigated, PD 102807 interacted with muscarinic receptors in a manner typical of a simple competitive antagonist. These data provide additional evidence that PD 102807 is a M4-receptor preferring antagonist and that this compound can discriminate the striatal muscarinic receptors inhibiting DA D1 receptor activity from the cortical receptors mediating the potentiation of CRH receptor signalling and the inhibition of Ca2+/CaM-stimulated adenylyl cyclase activity.     

Ligand specificity and affinity of BT-R1, the Bacillus thuringiensis Cry1A toxin receptor from Manduca sexta, expressed in mammalian and insect cell cultures.

The Manduca sexta receptor for the Bacillus thuringiensis Cry1Aa, Cry1Ab, and Cry1Ac toxins, BT-R1, has been expressed in heterologous cell culture, and its ligand binding characteristics have been determined. When transfected with the BT-R1 cDNA, insect and mammalian cell cultures produce a binding protein of approximately 195 kDa, in contrast to natural BT-R1 from M. sexia, which has an apparent molecular weight of 210 kDa. Transfection of cultured Spodoptera frugiperda cells with the BT-R1 cDNA imparts Cry1A-specific high-affinity binding activity typical of membranes prepared from larval M. sexta midguts. Competition assays with BT-R1 prepared from larval M. sexta midguts and transiently expressed in cell culture reveal virtually identical affinities for the Cry1Aa, Cry1Ab, and Cry1Ac toxins, clearly demonstrating the absolute specificity of the receptor for toxins of the lepidopteran-specific Cry1A family. BT-R1 therefore remains the only M. sexta Cry1A binding protein to be purified, cloned, and functionally expressed in heterologous cell culture, and for the first time, we are able to correlate the Cry1Aa, Cry1Ab, and Cry1Ac toxin sensitivities of M. sexta to the identity and ligand binding characteristics of a single midgut receptor molecule.     

Design, synthesis, and biological evaluation of pirenzepine analogs bearing a 1,2-cyclohexanediamine and perhydroquinoxaline units in exchange for the piperazine ring as antimuscarinics

Pirenzepine (2) is one of the most selective muscarinic M(1) versus M(2) receptor antagonists known. A series of 2 analogs, in which the piperazyl moiety was replaced by a cis- and trans-cyclohexane-1,2-diamine (3-6) or a trans- and cis-perhydroquinoxaline rings (7 and 8) were prepared, with the aim to investigate the role of the piperazine ring of 2 in the interaction with the muscarinic receptors. The structural change leading to compounds 3-6 abolished in binding assays the muscarinic M(1)/M(2) selectivity of 2, due to an increased M(2) affinity. Rather, compounds 3-6 displayed a reversed selectivity showing more affinity at the muscarinic M(2) receptor than at all the other subtypes tested.     

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

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

Effects of Geissoschizine Methyl Ether, an Indole Alkaloid in Uncaria Hook, a Constituent of Yokukansan, on Human Recombinant Serotonin7 Receptor

Effects of seven alkaloids, geissoschizine methyl ether (GM), hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, corynoxeine and isocorynoxeine, in Uncaria hook, a constituent of the kampo medicine yokukansan, on serotonin₇ (5-HT₇) receptor were investigated using Chinese hamster ovary (CHO) cell membranes and human embryonic kidney 293 (HEK293) cells stably expressing the human recombinant 5-HT₇ receptor. A competitive binding assay using CHO membranes showed that GM (IC₅₀ = 0.034 μM) more strongly inhibited the binding of the radioligand [³H] LSD to 5-HT₇ receptor than the other alkaloids, suggesting that GM is bound to 5-HT₇ receptor. Agonistic/antagonistic effects of GM (1–50 μM) on the receptor were evaluated by measuring intracellular cAMP levels in HEK239 cells. GM (IC₅₀ = 6.0 μM) inhibited 5-HT-induced cAMP production in a concentration-dependent manner, as well as the specific 5-HT₇ receptor antagonist SB-269970 (0.1–1 μM). However, GM did not induce intracellular cAMP production as 5-HT did. These results suggest that GM has an antagonistic effect on 5-HT₇ receptor.     

Solubilization, separation, and partial characterization of histamine H1 and H2 receptors from calf thymocyte membranes.

Histamine membrane receptors are defined as either H1 (blocked by diphenhydramine-like antagonists) or H2 (blocked by cimetidine-like agents). We now report the solubilization, separation, and partial characterization of specific H1 and H2 membrane receptors from calf thymocytes. Membrane fragments were incubated with [3H]histamine either alone or with unlabeled histamine, diphenhydramine, or cimetidine. Maximal specific binding occurred with incubation at 37 degrees C for 2 h at a concentration of 5 x 10(-6) M [3H]histamine. Labeled receptors were solubilized from membranes with 0.3 M KCl and 1% Nonidet 40. Chromatography of the solubilized labeled receptors on ion exchange columns revealed two classes of receptor. One class bound to DEAE-cellulose and eluted as a sharp peak at 0.15 M NaCl/Pi. The other bound to phosphocellulose and eluted as a sharp peak at 0.55 M NaCl/Pi. Initial incubation of the membranes in the presence of the H1 receptor antagonist diphenhydramine virtually abolished the DEAE-cellulose peak, while incubation with cimetidine, the H2 receptor antagonist, blocked the phosphocellulose peak. We conclude that H1 and H2 histamine receptors are physically separable and can be defined by their ability to bind to either DEAE-cellulose or phosphocellulose.     

Induction of preputium eversion by peptides, serotonin receptor antagonists, and selective serotonin reuptake inhibitors in Biomphalaria glabrata

Abstract. Eversion of the preputium is one of the initial steps in the male copulatory behavior of freshwater pulmonates. Previous experiments have shown that serotonergic mechanisms are involved in eversion in the snail Biomphalaria glabrata because the vertebrate 5-HT₁ receptor antagonist methiothepin caused long-lasting eversion in a dose-dependent manner. In this study, we tested a variety of serotonergic receptor ligands, bioactive peptides, and selective serotonin reuptake inhibitors (SSRIs) for their ability to induce preputium eversion in B . glabrata in order to elucidate the physiological mechanism of eversion. Of 15 compounds tested, five significantly induced preputium eversion: the serotonin receptor antagonists methiothepin (1 and 10 μM; p<0.0001), cyproheptadine (1–10 μM; p<0.007–0.0001), and mianserin (5–50 μM; p<0.01–0.001), the molluscan cardioactive peptide FMRFamide (10 and 50 μM; p<0.0002–0.0001), and the SSRI fluoxetine (=Prozac, 10–100 μM; p<0.0003–0.0001). Serotonin itself neither induced eversion nor blocked methiothepin-induced eversion. This suggests that fluoxetine is not acting as an SSRI, but potentially as a receptor ligand. These preliminary data shed light on the possible physiological mechanism of preputium eversion in B . glabrata and suggest similarity with that of the model freshwater gastropod Lymnaea stagnalis .     

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal, airway and urinary bladder smooth muscle

Both M(2) and M(3) muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M(3) receptors interact with G(q) to trigger phosphoinositide hydrolysis, Ca(2+) mobilization and a direct contractile response. In contrast, M(2) receptors interact with G(i) and G(o) to inhibit adenylyl cyclase and Ca(2+)-activated K(+) channels and to potentiate a Ca(2+)-dependent, nonselective cation conductance. Ultimately, these mechanisms lead to the prediction that the influence of the M(2) receptor on contraction should be conditional upon mobilization of Ca(2+) by another receptor such as the M(3). Mathematical modeling studies of these mechanisms show that the competitive antagonism of a muscarinic response mediated through activation of both M(2) and M(3) receptors should resemble the profile of the directly acting receptor (i.e., the M(3)) and not that of the conditionally acting receptor (i.e., the M(2)). Using a combination of pharmacological and genetic approaches, we have identified two mechanisms for the M(2) receptor in contraction: 1) a high potency inhibition of the relaxation elicited by agents that increase cytosolic cAMP and 2) a low potency potentiation of contractions elicited by the M(3) receptor. The latter mechanism may be involved in muscarinic agonist-mediated heterologous desensitization of smooth muscle, which requires activation of both M(2) and M(3) receptors.     

The effects of LAMP1 and LAMP3 on M180 amelogenin uptake, localization and amelogenin mRNA induction by amelogenin protein

We previously demonstrated that the uptake of M180 amelogenin protein in dental epithelial cells (HAT-7) results in increased levels of amelogenin mRNA through enhanced mRNA stabilization. To determine the processes involved in the uptake of extracellular M180 amelogenin by cells and in amelogenin intracellular trafficking in the amelogenin protein-mediated amelogenin mRNA expression pathway, we investigated the effects of LAMP1 and LAMP3, which are candidate M180 amelogenin receptors, on M180 amelogenin uptake, localization and amelogenin mRNA induction by amelogenin protein, using anti-LAMP-1 and anti-LAMP-3 antibodies and siRNA analysis. The results indicate that LAMP3 blocking by anti-LAMP-3 decreases M180 amelogenin uptake, but does not affect amelogenin mRNA induction by amelogenin protein, suggesting that LAMP3 is related to amelogenin degradation. Down-regulation by siRNA of LAMP1, which is the receptor for small amelogenin protein (LRAP), does not affect M180 amelogenin uptake, localization or amelogenin mRNA induction by amelogenin protein. Thus, while LAMP1 is the specific receptor for LRAP, it is not a receptor for M180 amelogenin. These findings will aid further research into the understanding of M180 amelogenin function and expression.