Isoform specificity of P2X2 purinergic receptor C-terminus binding to tubulin.
Adenosine triphosphate (ATP) and other nucleotides can be released in the central and peripheral nervous systems and act as neurotransmitters/neuromodulators. They can activate G-protein coupled receptors and ligand-gated ion channels, which are present throughout the central nervous system (CNS). P2X2 is one of seven known ion channels gated by ATP, and is characterized by having two transmembrane domains, a large extracellular loop and intracellular N- and C-termini. Recently, work from several laboratories has shown that neurotransmitter receptors can interact with other proteins thereby changing their functional attributes. More specifically, it was demonstrated that P2X2 binds beta-tubulin. Our goal was to investigate this interaction, by comparing P2X2 with a naturally occurring splicing variant named P2X2b. These isoforms differ in their C-terminal regions which contain the proposed beta-tubulin-binding domain. Indeed we were able to demonstrate that only the long variant P2X2 binds beta-tubulin I in various biochemical assays. In addition, this interaction can be direct since it also occurred when the P2X2 C-terminus was exposed to purified brain tubulin. When expressed in heterologous cells, P2X2 interacted with beta-tubulin I while present on the outer membrane, as demonstrated by biotinylation of surface proteins. Therefore, the present data strongly support a functional interaction between an ATP-gated channel and the cytoskeleton. Moreover, we show a biochemical difference between the splicing alternatives that might account for novel distinct functional roles.
Guimaraes
Marilia Z P
MZ
Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA. marilia@farmaco.ufrj.br
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000378:metabolism*
D020033:Protein Isoforms; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D014404:Tubulin