International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

F52 Identifying Interacting Partners to the Mouse Myosin VIIA Protein Involved in Genetic Deafness

Penio Todorov1, Rachel E. Hardisty1, Karen P. Steel2 and Steve D.M. Brown1. 1MRC Mouse Genome Centre and Mammalian Genetics Unit, Harwell, OX11, ORD, UK; 2MRC Institute of Hearing Research, University Park, Nottingham, NG7 2RD, UK

The shaker1 (Myo7a) mouse deafness locus encodes an unconventional myosin gene - myosin VIIA. The myosin VIIA protein is expressed in hair cells in the cochlea where it is thought to be involved in stereocilia positioning at the apical surface of the hair cells. Like all myosins, myosin VIIA possesses a conserved motor or head domain which contains ATP- and actin-binding sites. However, the structure of the tail domain is unique for each myosin class and is an important determinant in defining the specific cargo functions of any particular myosin. The myosin VIIA tail has a number of domains including a region of shared homology with myosin IV, a region of homology to the band 4.1 family as well as a putative SH3 domain (Mburu et al. 1997 Genes and Function 1: 191). Intriguingly the region of shared homology with myosin IV demonstrates very significant sequence similarity to the tail domain of a calmodulin-binding kinesin protein found in plants. Nevertheless, the function of the myosin VIIA tail domain is still unknown. In an attempt to further characterise myosin VIIA function we set out to identify molecule(s) that specifically associate with myosin VIIA. We have demonstrated that 17kDa and 55kDa proteins from mouse kidney co-purify with myosin VIIA on affinity columns with immobilised anti-myosin VIIA antibody. This antibody was raised against a peptide corresponding to the C-terminal 17 amino acids of the myosin VIIA tail. Amino terminal sequencing and immunoblotting analysis identified the 17kDa myosin VIIA associated protein as calmodulin. Myosin VIIA can be co-immunoprecipitated from kidney homogenate using anti-calmodulin antibody confirming the strong association between calmodulin and myosin VIIA. Myosin VIIA can also be co-purified from kidney using an affinity-purified polyclonal antibody raised against the 55kDa protein. N-terminal sequence analysis indicated that the 55kDa protein is novel and further characterisation is underway. The 55kDa antibody is also being used for immunostaining on cochlear sections. The characterisation of the interaction of calmodulin and 55kDa protein with myosin VIIA may provide further insights into the hair cell function of myosin VIIA.

 


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