Dual signaling regulated by Calcyon, a D1 dopamine receptor interacting protein

Science

Volumn 287, Issue 5458, 2000, Pages 1660-1664

  Lezcano, Nelson ^a   Mrzljak, Ladislav ^b   Eubanks, Steven ^a   Levenson, Robert ^c   Goldman Rakic, Patricia ^b   Bergson, Clare ^a  

^a MEDICAL COLLEGE OF GEORGIA   (United States)

^b YALE UNIVERSITY SCHOOL OF MEDICINE   (United States)

^c PENN STATE COLLEGE OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CALCYON; DOPAMINE 1 RECEPTOR; GUANINE NUCLEOTIDE BINDING PROTEIN; MEMBRANE PROTEIN; UNCLASSIFIED DRUG;

ARTICLE; CALCIUM SIGNALING; CALCIUM TRANSPORT; CELL; COGNITION; DENDRITIC SPINE; MOLECULAR INTERACTION; NONHUMAN; PREFRONTAL CORTEX; PRIORITY JOURNAL; PYRAMIDAL NERVE CELL; RECEPTOR BINDING; RECEPTOR OCCUPANCY; SIGNAL TRANSDUCTION; YEAST;

AMINO ACID SEQUENCE; ANIMALS; BENZAZEPINES; BRAIN; CALCIUM; CALCIUM SIGNALING; CELL LINE; CYCLIC AMP; DENDRITES; DOPAMINE AGONISTS; FEMALE; HETEROTRIMERIC GTP-BINDING PROTEINS; HUMANS; MACACA MULATTA; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; PREFRONTAL CORTEX; PYRAMIDAL CELLS; RABBITS; RECEPTOR CROSS-TALK; RECEPTORS, DOPAMINE D1; RECEPTORS, NEUROTRANSMITTER; RECOMBINANT FUSION PROTEINS; SIGNAL TRANSDUCTION; TWO-HYBRID SYSTEM TECHNIQUES;

EID: 0034048720     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5458.1660     Document Type: Article

References (43)

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11. GenBank accession number for calcyon is AF225903. Supplemental data are available at www.sciencemag. org/feature/data/1044333.shl
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17. 3 (pH 11) or 10 mM Hepes (pH 7.4) and 5 mM EDTA buffer containing 500 mM NaCl, 6 M urea, or 100 mM Nal. For immunoblotting, proteins in loading buffer [U. K. Laemmli, Nature 227, 680 (1970)] were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride sheets [H. Towbin, T. Staehelin, J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979)]. Molecular mass was determined relative to mobility of Perfect protein markers or prestained broad range markers. Immunoblots were incubated with rabbit antibodies to calcyon (1:100) followed by horseradish peroxidase (HRP)-conjugated antibodies to rabbit (antirabbit antibodies) (1:25,000).
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19. Perfusion and preparation of brain tissue from adult macaque monkeys (Macaca mulatta) were carried out as described [L. Mrzljak, A. I. Levey, S. Belcher, P. S. Goldman-Rakic, J. Comp. Neurol. 390, 112 (1998)]. For double-labeling experiments, 40-μm tissue sections were incubated with a cocktail of rat D1 monoclonal antibody (13) and rabbit calcyon antibodies for 48 hours at 4°C, washed, and incubated with Cy3-conjugated antirabbit or FITC-conjugated secondary antirat antibodies (12). Sections were viewed with a Molecular Dynamics confocal argon/krypton laser and Nikon Diaphot 200 microscope, and data were collected with Molecular Dynamics Image Space software and analyzed as a Ray model projection of 30 1-μm sections. For electron microscope analysis, sections were processed by the preembedding silver intensified immunogold method with goat antirabbit IgG coupled to 1.4-nm gold particles.
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23. 365-446 were generated by polymerase chain reaction (PCR), subcloned into pGBT9 (5), verified by DNA sequencing with an ABI automated DNA sequencer, and then tested for interaction with pACT-calcyon in Y190 (6). Positive two-hybrid interactions were visibly blue within 2 to 6 hours at 30°C.
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42. Amino acid residues are abbreviated as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
43. Supported by P50 MH44866 (P.G.-R.), NARSAD (C.B.), and National Institute of Mental Health grant MH56608 (C.B.). We thank W. Konigsberg (Yale University), J. Robishaw [Pennsylvania State University (PSU)], D. Lewis, and N. Lambert [Medical College of Georgia (MCG)] for helpful discussions; A. Hopper, G. Vaduva (PSU), J. A. Johnson, and W. Bollag (MCG) for generously supplying reagents; and the Imaging Core Facility (MCG) for technical assistance.