The long term objective of this study is to understand the functioning of receptors so as to be able to design new artificial receptors for therapies for diseases in which receptor functions fail or need better timing. To achieve this objective several important steps must be taken and these are the specific aims of this proposal. The first is to understand the mechanism of transmembrane signaling. Tools for this purpose are being developed and applied to the aspartate receptor that can then be applied to more complex receptors.
A second aim i s to alter the specificity of a receptor so that unnatural compounds can initiate a signaling system. This two fold program is the prologue to the third aim of making chimeric receptors that can be turned on and off by artificial compounds according to the timing of the experimenter. As a model for such systems, the aspartate-insulin chimeric receptor will be studied, in particular its translocation to the desired location, because ultimately we want the receptor in the outer membrane. Finally the signaling pathway for chemotaxis will be explored to see whether it is present in mammalian cells. Because the phosphate groups that are involved in the bacterial signaling pathway are histidine phosphate and carboxyl phosphate in contrast to the serine, threonine and tyrosine phosphates of the known mammalian pathways we will make antibodies against histidine phosphate, identifying the proteins by Western blots and sequencing them. Carboxyl phosphate are less stable but may then be looked for by LiBH 4 reduction and sequencing.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK009765-31A2
Application #
2015938
Study Section
Biochemistry Study Section (BIO)
Program Officer
Margolis, Ronald N
Project Start
1977-12-01
Project End
1999-05-31
Budget Start
1997-06-01
Budget End
1998-05-31
Support Year
31
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Jeffery, C J; Koshland Jr, D E (1994) A single hydrophobic to hydrophobic substitution in the transmembrane domain impairs aspartate receptor function. Biochemistry 33:3457-63
Biemann, H P; Koshland Jr, D E (1994) Aspartate receptors of Escherichia coli and Salmonella typhimurium bind ligand with negative and half-of-the-sites cooperativity. Biochemistry 33:629-34
Shapiro, M J; Koshland Jr, D E (1994) Mutagenic studies of the interaction between the aspartate receptor and methyltransferase from Escherichia coli. J Biol Chem 269:11054-9
Morimoto, B H; Koshland Jr, D E (1994) Conditional activation of cAMP signal transduction by protein kinase C. The effect of phorbol esters on adenylyl cyclase in permeabilized and intact cells. J Biol Chem 269:4065-9
Cheever, L; Koshland Jr, D E (1994) Habituation of neurosecretory responses to extracellular ATP in PC12 cells. J Neurosci 14:4831-8
Danielson, M A; Biemann, H P; Koshland Jr, D E et al. (1994) Attractant- and disulfide-induced conformational changes in the ligand binding domain of the chemotaxis aspartate receptor: a 19F NMR study. Biochemistry 33:6100-9
Jeffery, C J; Koshland Jr, D E (1993) Three-dimensional structural model of the serine receptor ligand-binding domain. Protein Sci 2:559-66
Yeh, J I; Biemann, H P; Pandit, J et al. (1993) The three-dimensional structure of the ligand-binding domain of a wild-type bacterial chemotaxis receptor. Structural comparison to the cross-linked mutant forms and conformational changes upon ligand binding. J Biol Chem 268:9787-92
Stoddard, B L; Koshland Jr, D E (1993) Molecular recognition analyzed by docking simulations: the aspartate receptor and isocitrate dehydrogenase from Escherichia coli. Proc Natl Acad Sci U S A 90:1146-53
Martin, P T; Chung, B T; Koshland Jr, D E (1993) Regulation of neurosecretory habituation by cAMP. Role of adaptation of cAMP signals. Eur J Biochem 217:259-65

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