News and Publications

Functional Genomics and Signal Transduction

S. Kamisue,* I. Kuwabara, F.-T. Liu,** H. Maruyama, I. Maruyama, Y. Miyatani, T. Moriki, M. Niwa, M. Shima,* A. Yoshioka*

* Nara Medical University, Nara, Japan
** La Jolla Institute for Allergy and Immunology, San Diego, CA

A variety of extracellular information ranging from ion concentration to cell-cell contact modify cellular activities such as differentiation and motility. The external signal is often received by transmembrane receptors and transmitted to the inside of the cell through various molecular cascades in which protein-protein interactions and second messengers play pivotal roles. We are using molecular genetic techniques to understand the molecular mechanisms that underlie such signal transduction pathways.

We have developed novel expression vectors, based on bacteriophage , that can produce foreign proteins as fusions on the surface of the phage particle. These surface-expression systems revolutionize screening of cDNA libraries by affinity selection, because macromolecules immobilized on solid matrices such as microtiter wells and paramagnetic beads can be used as probes. In the past year, we used the vectors to map antibody epitopes and to determine protein domains involved in macromolecular interactions during signal transduction. We are using these systems to determine protein-protein and protein-DNA interactions. Worldwide genome sequencing projects are determining all the genes of many organisms. The phage display should be a powerful tool in functional genomics for identifying macromolecular interactions.

We are also trying to understand a signal transduction involved in neurotransmission. Regulation of the release of neurotransmitters at nerve terminals is a key process in many functions of the nervous system, from neuronal development to memory formation. Neurotransmission is a complex process involving a number of distinct proteins. One protein, UNC-13 of Caenorhabditis elegans, discovered in our laboratory, plays a major role as a calcium sensor in the process. Our aim is to elucidate how release of neurotransmitters is regulated by UNC-13 and to discover other proteins involved in neurotransmission.

Immunohistochemical studies with specific antibodies to UNC-13 indicated that UNC-13 is localized at presynaptic terminal membranes of synapses. To elucidate the novel signal transduction pathway in neurotransmission, we are using genetic studies and the phage surface-expression systems we developed to search for effector proteins that directly interact with UNC-13.

To understand the molecular mechanism that underlies transmembrane signal transduction, we analyzed the aspartate receptor Tar. This receptor is a component of the chemotaxis signal transduction pathway and acts as a sensor for attractant and repellent. To understand how the external signals are transmitted to the inside of the cell, we used a novel genetic approach to replace the transmembrane segment with random peptides and searched for clones that could transmit the signal.

The results revealed an essential structure of the segment for the function: an -helix with 3 distinct faces. Subsequent disulfide cross-linking of the faces suggests that dynamic rotation or twist of the segment is frozen at 1 face of the helix by the attractant binding and at another face by the repellent binding. This simple dynamic model of transmembrane signaling is being tested for other cell-surface receptors such as those for epidermal growth factor and insulin-like growth factor.

PUBLICATIONS

Kuwabara, I., Maruyama, I.N. Epitope mapping by phage display. Jpn. J. Thromb. Hemost. 9:166, 1998.