Connexins are transmembrane proteins that form aqueous channels, known as gap junctions, which directly interconnect the cytoplasms of adjacent cells and permit the exchange of small molecules. Gap junctions play important roles in numerous functions, including cardiac and smooth muscle function, electrotonic transmission in nerves, metabolic cooperation in avascular organs such as the lens, and in development. Recently, the disturbance of normal connexin function has been discovered in specific human diseases, such as Charcot-Marie-Tooth peripheral neuropathy (connexin32) and hereditary non-syndromic sensorineural deafness (connexin26). It has also been postulated that the disruption of gap junctional communication may lead to a loss of growth control that contributes to the development of human cancer. In this study, we will investigate the mechanism(s) by which tyrosine kinase oncoproteins and growth factors regulate the functions) of connexin43 by phosphorylation. We will also study the regulation of connexin43 function by putative mechanism(s) involving the interaction of Cx43 with cellular proteins.
Specific Aim 1 will elucidate the role that phosphorylation plays in the regulation of connexin43 by tyrosine kinase oncoproteins and growth factors. Site directed mutagenesis and dye transfer studies will clarify the mechanism(s) by which tyrosine and serine phosphorylation of connexin43 induced by v-Src and the EGF receptor disrupts connexin43 channel permeability.
Specific Aim 2 will establish if the phosphorylation-induced disruption of connexin43 channels occurs by a """"""""particle-receptor"""""""" mechanism. This work will be accomplished by the microinjection of mutant connexin43 genes and peptides into Xenopus oocytes.
Specific Aim 3 will determine the mechanism(s) and functional significance of the interactions between connexin43 and ZO-1, a tight and adherans junction associated protein, and CIP7, a novel cysteine-rich protein. These studies will utilize mutants of connexin43, ZO-1, and CIP7, together with appropriate antibodies, to study the regulation of Cx43 function, subcellular localization, and phosphorylation. We will utilize techniques such as the microinjection of fluorescent dye, confocal and electron microscopy, co-immunoprecipitation and other biochemical procedures.
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