The University of Melbourne
	Department of Surgery
	Royal Melbourne Hospital and Western Hospital

Molecular Neurobiology Laboratory
Unit Head: Dr. Chris Hovens

Research

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Background

In order for living organisms to be able to grow, develop and maintain their normal physiological state they need to be able to co-ordinate the functions of different organs and tissues.
To do this they need to have mechanisms for signaling or sending information to cells. The cells themselves that make up all tissues and organs, have developed complex internal signaling networks that allow them to respond to body signals.
This signaling network is made up of proteins that can bind to one another and activate their functions.
In this way growth signals are transmitted into the interior of the cells.

In diseases such as cancer, tumors form because this communication process has gone awry. Cells inappropriately receive signals telling them to keep growing and dividing.
In order to be able to eventually develop new drugs that can block this process in a controlled way we need to know all the proteins that cells use to transmit and receive growth signals.
One class of proteins that are now known to be very important in the growth process and the development of tumors are cell surface receptors called Receptor Tyrosine Kinases or RTKs for short.
As yet we still do not know all the proteins that can bind and therefore communicate with this important class of proteins.
We have recently discovered that a protein called AF-6 can bind to some of the RTK proteins known as Eph receptors. Eph receptors are highly expressed in the brain and are important in axon pathway selection during neurogenesis.

Importantly, we have also discovered that AF-6 only binds to or communicates with RTKs that are already activated. That is, RTKs that are already giving a strong growth signal to cells.
It now seems likely that AF-6 will be an important component in the signaling mediated by this important class of receptors.

Finding out more about AF-6 and how it functions in cellular communication networks could improve our understanding of normal growth and more importantly of abnormal growth processes that lead to cancer.

Aims
Our aim is to understand the function of two proteins important in the transmission of growth signals. One of these is AF-6, the other is NF2. NF2 is a tumor suppressor protein whose loss or absence is clearly linked to the development of certain types of brain tumors.

Projects
The Laboratory is currently pursuing three main projects.

-The role of the AF-6 protein in signal transduction mediated by the brain Eph Receptor Tyrosine Kinases and its possible involvement in molecular pathways involved in memory and learning and in tumorigenesis.

-Molecular analysis of the tumor suppressor protein, NF2 (merlin) whose loss leads to the development of a variety of CNS tumors. We aim to identify the direct molecular targets of the NF2 protein and thereby attempt to understand how its loss leads to unrestricted cellular growth.
Neurofibromatosis type 2 (NF-2) is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. The NF-2 protein, also known as merlin or schwannomin is a recently cloned tumor suppressor and is mutated or inactivated in most schwannomas and meningiomas.
Homology analysis indicates that the NF-2 protein is most closely related to members of the protein 4.1 superfamily especially ezrin, radixin and moesin, the ERM proteins. ERM proteins link membrane proteins to the cytoskeleton. It has been speculated that disruption of a similar membrane-linking role for NF-2 is involved in the development of tumors. To date, almost nothing is known concerning potential interacting cellular proteins of NF-2 or of the organization and role of its functional domains.
We propose to perform a stuctural analysis of the human NF-2 protein to identify functional domains important for the sub-membranous localization of the protein using molecular biological and confocal microscopy techniques.
We will also perform genetic screens using specific functional domains of the human NF-2 protein in attempt to identify interacting cellular partner proteins.
Identifying such cellular proteins will be an important first step to identifying the normal cellular role of NF-2/merlin/schwanommin and potentially provide target proteins for possible future therapeutic approaches to treat the disease.

-Projects aimed at developing novel drug treatment regimens to re-establish NF2 protein expression in sporadic meningiomas and acoustic schwannoma cells.

Personnel

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Dr. Christopher Hovens, Ph.D.
- Laboratory Head email: c.hovens@surgeryrmh.unimelb.edu.au

Phone: 9342 7704; Fax: 9347 6488

Dr. James A. J. King, M.B., B.S. - Ph.D. student
Dr. Michael Buchert, Ph.D. - Visiting Fellow (from July 1999)

Research Support

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The Molecular Neurobiology Laboratory is grateful for the support of the following funding agencies:

• The Friends of The Royal Melbourne Hospital Neuroscience Foundation
• Swiss National Science Foundation (from July 1999)
• NHMRC of Australia, project grant

Recent Publications

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Refereed Publications:

1. Hovens CM, Stacker SA, Andres AC, Harpur AG, Ziemiecki A, Wilks AF. (1992) Ryk a Receptor Tyrosine Kinase-related molecule with unusual kinase domain motifs. Proc. Natl. Acad. Sci. U.S.A. 89:11818-11822.

2. Stacker SA, Hovens CM, Vitali A, Pritchard AM, Baker E, Sutherland RG, Wilks AF. (1993) Molecular Cloning and chromosomal localisation of the human homologue of a receptor related to tyrosine kinases (RYK). Oncogene 8:1347-1356.

3. Gough NM, Rakar S, Hovens CM, Wilks AF. (1995) Localisation of two mouse genes encoding the protein tyrosine kinase receptor-related protein RYK. Mammalian Genome 6:255-6.

4. Gstaiger M, Knoepfel L, Georgiev O, Schaffner W, Hovens CM. (1995) A B-cell coactivator of octamer-binding transcription factors. Nature 373:360-362.

5. Georgiev O, Bourquin JP, Gstaiger M, Knoepfel L, Schaffner W, Hovens CM. (1996) Two versatile eukaryotic expression vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins. Gene 168:165-167.

6. Schneider S, Buchert M, Hovens CM. (1996) A rapid, simplified in vitro assay of b-galactosidase from yeast. Biotechniques 20:961-962.

7. Gstaiger M, Hovens CM, Geogiev O, Knoepfel L, Schaffner W. (1996) BZLF-1 (Zebra, Zta) protein of Epstein -Barr virus selected in a yeast One-Hybrid system by binding to a consensus site in the IgH intronic enhancer: A role in immunoglobulin expression? Biological Chemistry 377:669-673.

8. Buchert M, Schneider S, Adams M, Hefti HP, Moelling K, Hovens CM. (1997) Useful vectors for the Two Hybrid system in mammalian cells. Biotechniques 23:396-402.

9. Schneider S, Georgiev O, Buchert M, Adams M, Moelling KM, Hovens CM. (1997) An epitope tagged eukaryotic/prokaryotic expression vector with positive selection of cloned inserts. Gene 197:337-341.

10. Schneider S, Buchert M, Adams M Moelling KM, Hovens CM. (1997) Dual translation cassettes permitting prokaryotic and vetebrate protein expression from the same vector. Technical Tips Online T01086.

11. Schneider S, Buchert M, Georgiev O, Catimel B, Halford M, Stacker SA, Baechi T, Moelling K, Hovens CM. (1999) Mutagenesis and selection of PDZ domains that bind new protein targets. Nature Biotechnology 17: 170-175

12. Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani E, Baechi T, Moelling K, Hovens CM. (1999) The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain. Journal of Cell Biology 144:361-371.