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1.
J Pharmacol Exp Ther ; 315(3): 1278-87, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16135699

ABSTRACT

Drugs that antagonize D2-like receptors are effective antipsychotics, but the debilitating movement disorder side effects associated with these drugs cannot be dissociated from dopamine receptor blockade. The "atypical" antipsychotics have a lower propensity to cause extrapyramidal symptoms (EPS), but the molecular basis for this is not fully understood nor is the impact of inverse agonism upon their clinical properties. Using a cell-based functional assay, we demonstrate that overexpression of Galphao induces constitutive activity in the human D2-like receptors (D2, D3, and D4). A large collection of typical and atypical antipsychotics was profiled for activity at these receptors. Virtually all were D2 and D3 inverse agonists, whereas none was D4 inverse agonist, although many were potent D4 antagonists. The inverse agonist activity of haloperidol at D2 and D3 receptors could be reversed by mesoridazine demonstrating that there were significant differences in the degrees of inverse agonism among the compounds tested. Aripiprazole and the principle active metabolite of clozapine NDMC [8-chloro-11-(1-piperazinyl)-5H-dibenzo [b,e] [1,4] diazepine] were identified as partial agonists at D2 and D3 receptors, although clozapine itself was an inverse agonist at these receptors. NDMC-induced functional responses could be reversed by clozapine. It is proposed that the low incidence of EPS associated with clozapine and aripiprazole used may be due, in part, to these partial agonist properties of NDMC and aripiprazole and that bypassing clozapine blockade through direct administration of NDMC to patients may provide superior antipsychotic efficacy.


Subject(s)
Antipsychotic Agents/metabolism , Clozapine/metabolism , Receptors, Dopamine D2/agonists , Receptors, Dopamine D3/agonists , Receptors, Dopamine D4/metabolism , Animals , Haloperidol/pharmacology , Humans , Mice , NIH 3T3 Cells , Pergolide/pharmacology , Plasmids , RGS Proteins/metabolism , Transfection
2.
Neuropharmacology ; 42(7): 929-40, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12069903

ABSTRACT

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.


Subject(s)
Methylhistamines/pharmacology , Receptors, Histamine H3/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Guinea Pigs , Humans , Molecular Sequence Data , Protein Isoforms/drug effects , Protein Isoforms/genetics , Protein Isoforms/physiology , RNA Splicing , Rats , Receptors, Histamine H3/drug effects , Receptors, Histamine H3/physiology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino Acid , Structure-Activity Relationship
3.
J Neurochem ; 69(2): 485-93, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9231706

ABSTRACT

Nine isoforms of the rat NMDAR1 receptor subunit have been previously identified, of which several have an alternatively spliced N-terminal insert believed to be important in proton sensitivity of the receptor. The cloning of the human homologues of NMDAR1-3b (hNMDA1-1) and NMDAR1-4b (hNMDA1-2), both bearing the insert, is reported here. A monoclonal antibody generated against the N-terminal region of these isoforms showed reactivity with at least two distinct human brain proteins of approximately 115 kDa. This antibody was further characterized by using a series of truncated fusion proteins and splice variants of NMDAR1 demonstrating its specific recognition of an epitope within the 21-amino acid N-terminal insert, encoded by exon 5. Western blot and immunocytochemical studies were performed to examine the expression of the exon 5-containing isoforms of the NMDAR1 subunit in both rat and human brain.


Subject(s)
Brain Chemistry , Cloning, Molecular , Exons , Receptors, N-Methyl-D-Aspartate/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Antibody Specificity , Blotting, Western , Epitope Mapping , Humans , Immunohistochemistry , Molecular Sequence Data , RNA, Messenger/analysis , Rats , Receptors, N-Methyl-D-Aspartate/analysis , Receptors, N-Methyl-D-Aspartate/chemistry , Recombinant Fusion Proteins/immunology , Sequence Homology
4.
Ann Neurol ; 41(6): 742-53, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9189035

ABSTRACT

Mutations of the presenilin PS1 and PS2 genes are closely linked to aggressive forms of early-onset (< 60 years) familial Alzheimer's disease. A highly specific monoclonal antibody was developed to identify and characterize the native PS1 protein. Western blot analyses revealed a predominant 32-kd immunoreactive polypeptide in a variety of samples, including PC12 cells transfected with human PS1 complementary DNA, brain biopsy specimens from demented patients, and postmortem samples of frontal neocortex from early-onset familial Alzheimer's disease cases (PS1 and PS2), late-onset sporadic Alzheimer's disease cases, and cases of other degenerative disorders. This truncated polypeptide contains the N-terminus of PS1 and appeared unchanged across cases. In 2 early-onset cases linked to missense mutations in the PS1 gene, a PS1 immunoreactive protein (approximately 49 kd) accumulated in the frontal cortex. This protein was similar in size to full-length PS1 protein present in transfected cells overexpressing PS1 complementary DNA, and in lymphocytes from an affected individual with a deletion of exon 9 of the PS1 gene, suggesting that mutations of the PS1 gene peturb the endoproteolytic processing of the protein. Immunohistochemical studies of control brains revealed that PS1 is expressed primarily in neurons, with the protein localized in the soma and dendritic processes. In contrast, PS1 showed striking localization to the neuropathology in early-onset familial Alzheimer's disease and sporadic Alzheimers' disease cases. PS1 immunoreactivity was present in the neuritic component of senile plaques as well as in neurofibrillary tangles. Localization of PS1 immunoreactivity in familial and sporadic Alzheimer's disease suggests that genetically heterogeneous forms of the disease share a common pathophysiology involving PS1 protein.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Membrane Proteins/metabolism , Adult , Aged , Animals , Blotting, Western , Brain/metabolism , Haplorhini , Humans , Immunohistochemistry , Middle Aged , PC12 Cells/metabolism , Presenilin-1 , Rats , Tissue Distribution , Transfection
5.
J Neurosci ; 17(6): 1971-80, 1997 Mar 15.
Article in English | MEDLINE | ID: mdl-9045726

ABSTRACT

Several genes have been implicated in the pathogenesis of early-onset familial Alzheimer's disease. A majority of the autosomal dominant cases are linked to recently identified mutations in the presenilin-1 gene on chromosome 14. The native presenilin-1 protein in primates has not been well characterized, and its precise localization is unknown. We have studied the native presenilin-1 protein in monkey brain and peripheral tissues by using a monoclonal antibody specific for the N-terminal domain of human presenilin-1. Western blots detect polypeptide species of approximately 49 and approximately 32 kDa from COS-7 and PC12 cells transfected with full-length human presenilin-1 cDNA and from in vitro translations of the normal human presenilin-1 mRNA. A 32 kDa polypeptide is detected in monkey peripheral tissues, with the highest expression in testis and lung. In all brain regions the 32 kDa band is the predominant form of presenilin-1, and it is found in particulate subfractions. Light microscopic immunocytochemistry reveals presenilin-1 staining in all brain regions, with the strongest labeling in neurons and neuropil. In addition, weaker immunoreactivity is also present in glia and blood vessels. Neuronal staining shows significant variability, with particularly intense labeling of certain cell types, including large neocortical and hippocampal pyramidal neurons, magnocellular basal forebrain neurons, brainstem motoneurons, and some populations of interneurons. By electron microscopic immunocytochemistry, highly selective presenilin-1 staining is seen on the cytoplasmic surfaces of membranous organelles, which suggest localization to the endoplasmic reticulum-Golgi intermediate compartment, a subdomain of the endoplasmic reticulum, and some coated transport vesicles.


Subject(s)
Brain/metabolism , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Animals , Brain/ultrastructure , Humans , Immunohistochemistry , Macaca , Microscopy, Electron , Presenilin-1
6.
J Biol Chem ; 271(47): 29746-51, 1996 Nov 22.
Article in English | MEDLINE | ID: mdl-8939910

ABSTRACT

Chemical cross-linking combined with site-directed mutagenesis was used to evaluate the role of extracellular cysteines and their positions relative to the binding site for the agonist bradykinin (BK) in the human BK B2 receptor. All extracellular cysteines, Cys20, Cys103, Cys184, and Cys277, in the receptor were mutated to serines, and single and double mutants were transfected into COS-7 cells. The Ser20 and Ser277 single mutants and the Ser20/Ser277 double mutant bound [3H]BK and the antagonist [3H]NPC17731 with pharmacological profiles identical to the wild-type B2 receptor. In contrast, the Ser103 and Ser184 single mutants were unable to bind either of the two radioligands. However, these mutants were still expressed as determined by immunoblotting with anti-B2 receptor antibodies. Previous studies on the bovine B2 receptor showed that bifunctional reagents, which are reactive to amines at one end and to free sulfhydryls in the opposite end, cross-link the N terminus of receptor-bound BK to a sulfhydryl in the receptor (Herzig, M. C. S., and Leeb-Lundberg, L. M. F. (1995) J. Biol. Chem. 270, 20591-20598). Here, we show that m-maleimidobenzoyl-N-hydroxysuccinimide ester and 1,5-difluoro-2, 4-dinitrobenzene cross-linked BK to the wild-type human B2 receptor and the Ser20 and Ser277 single mutant receptors, whereas these reagents were unable to cross-link BK to the Ser20/Ser277 double mutant. These results show that Cys103 and Cys184 are both required for expression of high affinity agonist and antagonist binding sites in the human B2 receptor, while Cys20 and Cys277 are not required. Furthermore, the results provide direct biochemical evidence that the N terminus of BK, when bound to the B2 receptor, is adjacent to Cys277 in extracellular domain 4 and Cys20 in extracellular domain 1 of the receptor.


Subject(s)
Bradykinin/chemistry , Cysteine/chemistry , Receptors, Bradykinin/metabolism , Animals , Bradykinin/metabolism , COS Cells , Cross-Linking Reagents , Humans , Mutagenesis, Site-Directed , Protein Binding , Radioligand Assay , Receptor, Bradykinin B2 , Receptors, Bradykinin/genetics , Tritium
7.
J Neurosci ; 16(7): 2179-90, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8601799

ABSTRACT

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C-terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT-immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.


Subject(s)
Brain/metabolism , Carrier Proteins/metabolism , Choline O-Acetyltransferase/metabolism , Membrane Transport Proteins , Synaptic Vesicles/physiology , Vesicular Transport Proteins , Acetylcholine/metabolism , Animals , Antibody Specificity , Base Sequence , Biological Transport/physiology , Brain/ultrastructure , Carrier Proteins/immunology , Cholinergic Fibers/metabolism , Cholinergic Fibers/ultrastructure , Gene Expression/physiology , Immunoblotting , Immunohistochemistry , Microscopy, Immunoelectron , Molecular Sequence Data , Rabbits , Rats , Synaptic Transmission/physiology , Vesicular Acetylcholine Transport Proteins
8.
Proc Natl Acad Sci U S A ; 92(19): 8710-4, 1995 Sep 12.
Article in English | MEDLINE | ID: mdl-7568002

ABSTRACT

The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.


Subject(s)
Brain Chemistry , Hematopoietic Stem Cells/chemistry , Huntington Disease , Lymphocytes/chemistry , Nerve Tissue Proteins/isolation & purification , Nuclear Proteins/isolation & purification , Animals , Brain/cytology , Cell Compartmentation , Cell Line , Cytosol/chemistry , Haplorhini , Humans , Huntingtin Protein , Immunohistochemistry , Microscopy, Immunoelectron , Nerve Tissue Proteins/immunology , Neurons/chemistry , Nuclear Proteins/immunology , Rats , Recombinant Fusion Proteins/immunology , Tissue Distribution
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