Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina.

The messenger RNA (mRNA) that encodes alpha subunit of the guanosine triphosphate-binding protein transducin (T alpha) and T alpha immunoreactivity were localized and measured in the rat retina during the light-dark cycle with in situ hybridization and immunohistochemistry. Both T alpha mRNA and T alpha immunoreactivity were observed only in photoreceptors. Within the photoreceptor T alpha mRNA was present primarily in the inner segments and to a lesser extent in the outer nuclear layer at all times during the day and night. However, the distribution of T alpha immunoreactivity varied profoundly with the light-dark cycle; during the day, T alpha immunoreactivity was highest in the inner segments, and at night the outer segments were more immunoreactive. The amounts of T alpha mRNA and T alpha immunoreactivity also depended on the light-dark cycle. Levels of T alpha mRNA were high immediately before and after lights on; levels were low for the rest of the light-dark cycle. During the day, T alpha immunoreactivity increased in the inner segments following the increase in T alpha mRNA. After the lights were turned off, T alpha immunoreactivity decreased in the inner segments and increased in the outer segments. Thus, it appears that T alpha is synthesized in the inner segments after a morning increase in T alpha mRNA. Newly synthesized T alpha remains in the inner segments until it is transported to the outer segments at night, where it may be involved in the increase in the sensitivity of photoreceptor rods at night.

Identification of a family of muscarinic acetylcholine receptor genes.

Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.

Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes.

The human and rat genes for a fifth muscarinic receptor have been cloned and expressed in mammalian cells. The 532 amino acid human protein has 89% sequence identity to the 531 amino acid rat protein and is most closely related to the m3 receptor. Both proteins are encoded by single exons. The receptor has intermediate affinity for pirenzepine and low affinity for AF-DX 116, and it increases metabolism of phosphatidylinositol when stimulated with carbachol. Expression of mRNA has yet to be observed in brain or selected peripheral tissues, suggesting that either it is substantially less abundant than m1-m4 or its distribution is quite different.

Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.

Transforming G protein-coupled receptors transduce potent mitogenic signals in NIH 3T3 cells independent on cAMP inhibition or conventional protein kinase C.

We have used the expression of human acetylcholine muscarinic receptor (mAChR) genes in NIH 3T3 cells as a model for dissecting the molecular basis of cellular transformation induced by G protein-coupled receptors. Those mAChR subtypes efficiently coupled to PIP2 hydrolysis (m1, m3 and m5) induced agonist-dependent cell transformation whereas those inhibiting adenylyl cyclase (m2, m4) lack transforming activity. In the present study, we demonstrate that in cells expressing m1 but not m2 mAChRs the cholinergic agonist (carbachol) is alone as potent a stimulant for DNA synthesis as platelet-derived growth factor (PDGF) or serum. Furthermore, induction of DNA synthesis is shown to correlate with activation of PIP2 hydrolysis but not with inhibition of adenylyl cyclase. We also examined the role of protein kinase C (PKC) in mitogenic signalling through m1 mAChRs, and found that NIH 3T3 cells express PKC-alpha and PCK-zeta as the only conventional or Ca(2+)-independent PKC isozyme, respectively. Prolonged treatment with TPA depleted cells of PKC-alpha but not of PKC-zeta. In TPA-treated NIH 3T3 cells, the mitogenic response to a subsequent stimulation with TPA was absolutely abolished, but the response to PDGF or serum was not. Moreover, PKC depletion did not decrease DNA synthesis induced by carbachol. We conclude that carbachol potently induces reinitiation of DNA synthesis through the activation of transforming mAChR subtypes, independently of inhibition of adenylyl cyclase and conventional PKCs.

The ras-related GTPase rac1 regulates a proliferative pathway selectively utilized by G-protein coupled receptors.

Ras and rac are each members of the superfamily of monomeric GTPases and both function as molecular switches to link cell-surface signals to intracellular responses. Using a novel assay of cellular proliferation called R-SAT (Receptor Selection and Amplification Technology), we examined the roles of ras and rac in mediating the proliferative responses to a variety of cell-surface receptors. Activated, wild-type and dominant-negative mutants of rac and ras were tested for their effects on cellular proliferation either alone or in combination with receptors. Activated rac (rac Q61L, henceforth rac*) and ras (ras G12V, henceforth ras*) each induced strong proliferative responses. Dominant-negative rac (rac T17N, henceforth rac(-)) dramatically suppressed proliferative responses to G-protein coupled receptors (GPCR's) including the m5 muscarinic receptor and the alpha1B adrenergic receptor. In contrast, rac(-) had little or no effect upon responses to the tyrosine kinase receptor TrkC, and only partially suppressed responses to the Janus kinase (JAK/STAT) linked granulocyte macrophage colony stimulating factor (GM-CSF) receptor. Dominant-negative ras (ras T17N, henceforth ras(-)) blocked the proliferative responses to all of the tested receptors. Compared to rac(-) and ras(-), wild-type rac and ras had only modest effects on the tested receptors. Overall these results demonstrate that rac mediates the proliferative effects of G-protein coupled receptors through a pathway that is distinct from the proliferative signaling pathway utilized by tyrosine kinase linked and JAK-linked receptors.

Localization and quantitation of opsin and transducin mRNAs in bovine retina by in situ hybridization histochemistry.

Oligodeoxynucleotide probes complementary to a portion of bovine opsin mRNA and transducin mRNA were used for in situ hybridization histochemistry. Within the retina, only photoreceptors expressed mRNAs detectable with these probes, and the majority of both mRNAs were in photoreceptor inner segments. More opsin mRNA was detected than transducin mRNA. In the inner segments 0.54 +/- 0.05 copies/micron3 of opsin mRNA and 0.34 +/- 0.05 copies/micron3 of transducin mRNA were detected. In the outer nuclear layer, 0.39 +/- 0.06 copies/micron3 of opsin mRNA and 0.27 +/- 0.04 copies/micron3 of transducin mRNA were detected.

The distribution of a dopamine D2 receptor mRNA in rat brain.

Based on the recently reported sequence of a dopamine D2 receptor cloned from rat brain, we prepared a series of cDNA probes to determine the distribution of mRNA encoding this receptor. Within the forebrain, D2 receptor mRNA is abundant in the caudate-putamen, accumbens nucleus and olfactory tubercle. Moderate to low levels of mRNA are observed in the medial habenular nucleus, diagonal band, lateral septal nucleus, claustrum, dorsal endopiriform nucleus, and entorhinal cortex. In the mesencephalon, D2 receptor mRNA is abundant within the substantia nigra, pars compacta, and the ventral tegmental area. Comparison of the distribution of the mRNA and ligand binding indicates that both presynaptic and postsynaptic D2 receptors of the nigrostriatal, mesolimbic and mesocortical pathways are derived from the same mRNA.

Localization of mRNAs encoding the alpha-subunits of signal-transducing G-proteins within rat brain and among peripheral tissues.

The sequence of the mRNAs which encode the alpha-subunits of the signal-transducing G-proteins Gs, Go and two forms of Gi (termed Gi1 and Gi2) have recently been reported. Based on rat sequences we prepared oligodeoxynucleotide probes for measurement of these mRNAs in rat brain and peripheral tissues. The relative abundance of these mRNA species in brain was Gs greater than Go approximately Gi2 greater than Gi1. The Gs and Gi2 mRNAs had somewhat lower levels in heart, kidney and liver than in brain, and Go and Gi1 mRNAs were not detected in the peripheral tissues. Using in situ hybridization we localized each of these mRNAs within slices of the rat brain. The patterns of distribution of Gs and Gi2 mRNA were very similar, but very different from that of Go and Gi1 mRNA. These data illustrate that receptor-effector coupling G-proteins are regionally specialized in their expression. This regional specialization may reflect a selective coupling of individual G-proteins with the various neurotransmitter receptors and effector pathways.

Identification of a small intracellular region of the muscarinic m3 receptor as a determinant of selective coupling to PI turnover.

Molecular cloning studies have demonstrated the existence of five different muscarinic receptors (m1-m5). While m1, m3 and m5 strongly couple to stimulation of phosphoinositide (PI) hydrolysis, m2 and m4 are more efficiently linked to inhibition of adenylate cyclase. The sequences of m1-m5 have a short segment at the N-terminal portion of the putative third cytoplasmic loop (i3) which is highly conserved among m1, m3 and m5, but different from the sequence which is well conserved among m2 and m4. To study the role of this region in conferring coupling selectivity, we constructed cDNAs encoding chimeric m2/m3 receptors. Transient expression of these receptor hybrids in COS-7 cells showed that a 17 amino acid segment at the N-terminal portion of i3 is a major determinant of how efficiently the different muscarinic receptors are coupled to PI hydrolysis.

Bacterial expression of human muscarinic receptor fusion proteins and generation of subtype-specific antisera.

A family of five muscarinic acetylcholine receptor genes (m1-m5) encode highly related proteins; however, for methodological reasons it has not been possible to detect the gene products individually. To develop antibody probes specific for the receptor subtypes, unique regions of m1-m5 cDNAs, corresponding to the third cytoplasmic (i3) loops, were subcloned into bacterial expression vectors and the fusion proteins expressed in E. coli were used to generate rabbit antisera. These antisera react specifically with the respective fusion proteins on immunoblots and selectively immunoprecipitate each of the native cloned receptors. Since the i3 loops are immunogenic and the epitopes in the cloned receptors are accessible to antibodies, this approach should be valuable for immunological studies of the native receptors.

The striatum and cerebral cortex express different muscarinic receptor mRNAs.

The existence of four distinct muscarinic acetylcholine receptor genes (m1-mr) has recently been demonstrated. cDNAs for three of these receptors have been cloned from brain (m1, m3, m4) and one from heart (m2). To gain some understanding of the physiological role of the brain muscarinic receptors, we mapped the distribution of their mRNAs in rat brain by in situ hybridization. These mRNAs are barely detectable in the hindbrain and cerebellum. Within forebrain, each mRNA has a strikingly different pattern of distribution. The highest levels of m1 mRNA are in the cerebral cortex and hippocampus followed by the striatum. m3 mRNA is also prominent in the cerebral cortex, but has very low levels in the striatum. Conversely, the levels of m4 mRNA are highest in the striatum. Since the cognitive effects of muscarinic drugs have been localized to the cerebral cortex and hippocampus, and their psychomotor effects to the striatum, these data suggest that the muscarinic receptors which subserve these responses may be different gene products. Finally, we show that these muscarinic receptors can be distinguished pharmacologically, suggesting that it may be possible to develop drugs for the selective treatment of the psychomotor vs cognitive difficulties of Parkinson's and Alzheimer's disease, respectively.

Constitutive activation of muscarinic receptors by the G-protein Gq.

In the absence of ligands, G-protein coupled receptors interconvert between active and inactive conformations. These conformations are stabilized by agonists and antagonists, respectively. Like agonists, G-proteins are thought to preferentially associate with receptors in the active conformation and should therefore be able to promote their formation in the absence of agonist. We show that over-expression of Gq induces constitutive activation of compatible muscarinic receptors and that this activity is blocked by muscarinic antagonists. Gq also increases the potency and efficacy of agonists. These results indicate that regulation of G-protein levels has a profound impact on receptor control of cellular physiology, even in the absence of agonist ligands.

Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor.

The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known H(3) agonists, while all agonists tested displayed increased potency at isoform 2 relative to isoform 1. Histamine, N(alpha)-methylhistamine, and R(-) and S(+)-alpha-methylhistamine are 16-23-fold more potent, while immepip and imetit are three to fivefold more potent. Antagonist experiments revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor activity, and identified a small number of antipsychotics that possess significant antagonist activity.

Annulated heterocyclic bioisosteres of norarecoline. Synthesis and molecular pharmacology at five recombinant human muscarinic acetylcholine receptors.

A series of O-alkylated analogs of 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO) were synthesized and characterized as ligands for muscarinic acetylcholine receptors (mAChRs). O-Methyl-THAO (4a), O-ethyl-THAO (4b), O-isopropyl-THAO (4c), and O-propargyl-THAO (4d) were shown to be potent inhibitors of the binding of tritiated quinuclidinyl benzilate (QNB), pirenzepine (PZ), and oxotremorine-M (Oxo-M) to tissue membrane preparations. In the [3H]-Oxo-M binding assay, receptor affinities in the low nanomolar range were measured for 4a (IC50 = 0.010 microM), 4b (IC50 = 0.003 microM), 4c (IC50 = 0.011 microM), and 4d (IC50 = 0.0008 microM). Pharmacological effects (EC50 or Ki values) and intrinsic activities (per cent of maximal carbachol responses) were determined using five recombinant human mAChRs (m1-m5) and the functional assay, receptor selection and amplification technology (R-SAT). Compound 4c antagonized carbachol-induced responses at m1, m3, and m5. With the exception of 4b, which was an antagonist at m5, 4a,b,d showed partial agonism at m1-m5 with very similar subtype selectivity (m2 > m4 > m1 > or = m3 > m5). Agonist index values for 4a-d, which were calculated from [3H]QNB (brain) and [3H]Oxo-M (brain) binding data, were shown to be predictive of pharmacologically determined intrinsic activities at m1-m5, the same rank order of intrinsic activity being observed at all five mAChRs (4a > 4d > 4b > 4c). It is concluded that within this class of high-affinity mAChR (m1-m5) ligands, containing secondary amino groups, minor changes of the bioisosteric ester alkyl groups have marked effects on potency and, in particular, intrinsic activity.

Design and synthesis of m1-selective muscarinic agonists: (R)-(-)-(Z)-1-Azabicyclo[2.2.1]heptan-3-one, O-(3-(3'-methoxyphenyl)-2-propynyl)oxime maleate (CI-1017), a functionally m1-selective muscarinic agonist.

The synthesis and SAR of a series of (Z)-(+/-)-1-azabicyclo[2.2. 1]heptan-3-one, O-(3-aryl-2-propynyl)oximes are described. The biochemistry and pharmacology of 24Z (PD 142505) and its enantiomers are highlighted. 24Z is functionally an m1-selective muscarinic agonist. Efficacy and m1 selectivity reside in the R enantiomer, (R)-24Z (CI-1017).

Selectivity profile of the novel muscarinic antagonist UH-AH 37 determined by the use of cloned receptors and isolated tissue preparations.

1. Functional in vitro experiments were carried out to determine the antimuscarinic potencies of the pirenzepine derivative UH-AH 37 (6-chloro-5,10-dihydro-5-[(1-methyl-4-piperidinyl)acetyl]-11H-dibenzo- [b,e] [1,4] diazepine-11-one hydrochloride) at M1 muscarinic receptors of rabbit vas deferens, M2 receptors of rat left atria and M3 receptors of rat ileum. Furthermore, N-[3H]-methylscopolamine competition binding experiments were performed to obtain its affinities for the five cloned human muscarinic receptors (m1-m5) stably expressed in CHO-K1 cells. Pirenzepine served as a reference drug throughout all experiments. 2. In all preparations used, UH-AH 37 interacted with muscarinic receptors in a fashion characteristic of a simple competitive antagonist. 3. In the functional studies, UH-AH 37, like pirenzepine, showed high affinity for M1 (pA2 8.49) and low affinity for M2 muscarinic receptors (pA2 6.63). In contrast to pirenzepine, UH-AH 37 also displayed high affinity for M3 receptors (pA2 8.04). 4. In agreement with its functional profile, UH-AH 37 bound with highest affinity to m1 (pKi 8.74) and with lowest affinity to m2 receptors (pKi 7.35). Moreover, it showed a 7 fold higher affinity for m3 (pKi 8.19) than for m2 receptors, whereas pirenzepine bound to both receptors with low affinities. 5. The binding affinity of UH-AH 37 for m4 and m5 receptors (pKi 8.32 for both receptors) was only ca. 2.5 fold lower than that for m1 receptors, while the corresponding affinity differences were 6 and 13 fold in case of pirenzepine. 6. In conclusion, the receptor selectivity profile of UH-AH 37 differs clearly from that of its parent compound, pirenzepine, in both functional and radioligand binding studies, the major characteristics being its pronounced M2 (m2)/M3 (m3) selectivity. UH-AH 37 thus represents a useful tool for the further pharmacological characterization of muscarinic receptor subtypes.

Allosteric regulation of cloned m1-m5 muscarinic receptor subtypes.

Allosteric regulation of [3H]N-methylscopolamine [( 3H]NMS) and [3H]quinuclidinyl benzilate [( 3H]QNB) dissociation from the m1-m5 muscarinic receptor subtypes was examined in transfected CHO-K1 cells. Half-times of dissociation of [3H]NMS from cell membranes (at 23 degrees) ranged from less than 5 min for the m2 subtype to more than 60 min for the m5 subtype. For [3H]QNB, half-times (at 37 degrees) ranged from 1 hr (m2) to almost 4 hr (m3). The presence of gallamine slowed the dissociation of [3H]NMS from all of the subtypes, with an order of potency of m2 greater than m4 greater than m1 greater than m3 greater than m5. Dissociation of [3H]QNB from m1 and m2 receptors was modulated by gallamine in the biphasic manner that we have described previously for cardiac receptors; that is, low concentrations (1-10 microM) of gallamine accelerated dissociation, while 1 mM gallamine slowed it. Verapamil slowed the dissociation of [3H]-QNB from the m2 receptor in a monophasic manner, while the action of d-tubocurarine was qualitatively similar to that of gallamine. The potency of gallamine in allosterically regulating the m2 receptor was inversely related to ionic strength. Inactivation of pertussis toxin-sensitive G proteins abolished the ability of guanine nucleotides to regulate agonist affinity at the m2 receptor, but had no effect on allosteric regulation of the m2 receptor. These findings indicate that susceptibility to allosteric regulation varies in a complex way across muscarinic receptor subtypes and according to the choice of ligand.

Constitutive activation of chimeric m2/m5 muscarinic receptors and delineation of G-protein coupling selectivity domains.

To derive structure/function relationships for muscarinic receptor/G-protein coupling, the m2 and m5 muscarinic receptors and a series of m2/m5 chimeras were tested for agonist binding and functional responses in a cellular proliferation/transformation assay. m5, which mediates stimulation of phosphatidylinositol turnover, displayed robust activity in the proliferation assay, whereas m2, which mediates inhibition of adenylyl cyclase, was inactive in the proliferation assay. Chimeras that contained m2 sequences in the i2 or i3 loops had impaired activity or were inactive, respectively. Chimeras that contained m2 segments reaching from the N-terminus to TM2, or from TM6 to the C-terminus, had enhanced activity relative to m5, and a chimera with both of these elements was constitutively activated.

Muscarinic acetylcholine receptor subtypes: localization and structure/function.

Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The distributions of these subtypes and their potential physiological roles are discussed. By use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall, these studies have shown that amino acids within the third cytoplasmic loop of the receptors define their selectivities for different G-proteins and that multiple discontinuous epitopes contribute to their selectivities for different ligands. The residues that contribute to ligand binding and G-protein coupling are described, as well as the implied structures of these functional domains.