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Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.

ABSTRACT: CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.

SUBMITTER: McCarthy EV

PROVIDER: S-EPMC2756885 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.

McCarthy Ellena V EV Baggs Julie E JE Geskes Jeanne M JM Hogenesch John B JB Green Carla B CB

Molecular and cellular biology 20090817 20

CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mut ...[more]

PMID: 19687303

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Project description:The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.

Dataset Information

Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.

Publications

Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.

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