The vertebrate neuromuscular junction is a prototypical synapse designed for rapid localized transmission of information from motor nerve to muscle via highly specialized pre- and postsynaptic structures. The postsynaptic membrane contains high density clusters of nicotinic acetylcholine receptor (AChR) positioned precisely opposite the branches of the presynaptic nerve terminal. These AChR clusters are critical to synapse function. The goal of this project is to clarify the molecular mechanisms underlying formation and maintenance of AChR clusters in the postsynaptic membrane of the neuromuscular junction. Specifically, this study will address the roles of the cytosolic 43kD and 87kD postsynaptic proteins (43k and 87k protein) in AChR clustering. When expressed in heterologous cells, 43k protein forms clusters at the cell surface, and can induce coclustering of AChRs when both are expressed. In addition, 43k protein can cluster each AChR subunit (alpha, beta, gamma, and delta) individually at the cell surface. Using a recently developed, highly efficient quail fibroblast (QT-6) transient transfection system, we will investigate the protein-protein interactions that are important for AChR clustering. First, the region of the alpha subunit that is responsible for its interaction with 43k protein will be identified by mutating specific sites within the alpha subunit, then coexpressing the mutant with 43k protein in QT-6 cells to test for clustering as assessed by immunofluorescence microscopy. This mutational analysis will be complemented by also testing chimeras in which a portion of the alpha subunit has been inserted into a non-clustering transmembrane protein to determine whether this alpha subunit region contains the 43k protein interaction site. Once this site has been identified in alpha subunit, homologous sites in the other subunit will be mutated to demonstrate homologous function. Second, the minimum number of 43k protein interaction sites required for clustering of a fully assembled pentameric AChR will be determined by coexpressing 43k protein with fully assembled receptors in which one or more of the 43k protein interaction sites has been inactivated by site-directed mutagenesis. Clustering of the mutant AChRs in the transfected cells will be determined as above. Finally, we will investigate the role of 87k protein in AChR clustering by isolating and characterizing a cDNA clone for mouse 87k protein and preparing 87k protein-specific polyclonal antibodies for use in expression studies. 87k protein will be coexpressed with 43k protein or with 43k protein and AChR to determine whether it colocalizes with AChR/43k protein clusters or alters the appearance of these clusters. Mutational analysis will then be carried out on 87k protein to determine which domains are responsible for its activity. These experiments will help to elucidate the molecular mechanisms underlying the formation and maintenance of high density AChR clusters at the neuromuscular junction. In addition, this study may help to define the molecular basis of some congenital myasthenic syndromes which are characterized by a deficiency in AChRs in the postsynaptic membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29NS034070-05
Application #
2891977
Study Section
Special Emphasis Panel (ZRG2-PHY (02))
Program Officer
Nichols, Paul L
Project Start
1995-05-01
Project End
2001-04-30
Budget Start
1999-05-01
Budget End
2001-04-30
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Upstate Medical University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210