Characterization of G-protein betagamma expression in inner ear.

Heterotrimeric guanine nucleotide binding proteins (G-proteins) are composed of a diverse set of alpha, beta, and gamma subunits, which couple cell surface receptors to intracellular effectors, such as adenylyl cyclase, phospholipase Cbeta, and ion channels. Both the Galpha and the Gbetagamma dimers mediate effector activity and are believed to contribute to the complexity of the signaling pathway. Molecular and immunocytochemical techniques were employed to determine diversity of Gbeta and Ggamma subunit expression in the murine inner ear. PCR-based assessment of lambdaZAP unidirectional cDNA libraries, representing the cochlea and inner ear hair cells, indicated all five known Gbeta subunits were present in the cochlea, while only a subset of Ggamma isoforms were found. New or novel G-protein beta and gamma subunits were not detected. cDNAs representing Gbeta1-4 and Ggamma2, Ggamma3, Ggamma5, Ggamma8olf subunit transcripts were isolated. In addition, cDNAs corresponding to the Gbeta5 and Ggamma11 isoforms exhibited restricted expression to inner and outer hair cells, respectively. Antisera specific for Gbeta3, Gbeta4, Ggamma3, Ggamma5 and Ggamma11 stained spiral ganglion and neurosensory hair cells. A unique finding was the variable topological distribution of Ggamma3 in the spiral ganglion cells along the cochlear axis. Collectively, our results demonstrate a complementary as well as differential distribution pattern for Gbeta and Ggamma isoforms exists in the inner ear. The co-localization of various G-protein isoforms within the same cell type suggests specific combinatorial Gbeta and Ggamma subunit associations may preferentially be formed. Thus, the detection of multiple subunits presumably reflects the extent of the functional diversity of inner ear signaling pathways and should provide specificity of G-protein mediated pathways.

Characterization of lineage restricted forms of a Xenopus CD45 homologue.

The leukocyte common antigen, also known as CD45, is a structurally heterogenous molecule ranging in molecular weight from 180 to 220 kDa. CD45 belongs to a family of high molecular weight, cell surface glycoproteins expressed on all hematopoietic lineages with the exception of mature erythrocytes. In higher vertebrates, the highly conserved cytoplasmic domain of CD45 exhibits protein tyrosine phosphatase activity and has been implicated in lymphocyte activation through dephosphorylation of critical tyrosine residues on substrates associated with signal transduction pathways. The monoclonal antibody CL21 recognizes a high molecular weight determinant expressed on the surface of Xenopus leukocytes which was postulated to be a CD45 homologue. In order to determine if lymphocyte subpopulations expressed different molecular weight variants, splenic B cells were identified and isolated on the basis of surface IgM and the CL21 determinant expressed by these cells was compared to the determinant expressed by thymocytes. Immunoprecipitation revealed that IgM + B cells expressed a 220 kDa molecular weight variant whereas thymocytes and IgM-cells expressed a 180 kDa variant. Bone marrow myeloid cells, isolated on the basis of light scatter properties, expressed a determinant which ranged from 150 to 160 kDa. Dephosphorylation experiments utilizing p-nitrophenyl phosphate, 32P-labeled Raytide [tyr(P)], or Kemptide [ser(P)] as substrates demonstrated that immunoprecipitated CL21 antigen exhibited tyrosine specific phosphatase activity which was inhibited by sodium orthovanadate. Thus, data based on the presence of enzymatic activity and lineage restricted molecular weight variants support the hypothesis that the CL21 determinant is the amphibian homologue of mammalian CD45, and suggest that both structural and functional elements of CD45 have been conserved during vertebrate evolution.