Covalent binding of guanine nucleotides to the CD3‐γ chain of the T cell receptor/CD3 complex

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Abstract
In a search for proteins involved in signal transduction through the T cell receptor (TcR/CD3 complex), a recently developed highly efficient method for labeling of nucleotide binding proteins in permeabilized cells was applied. Here, we report that human CD3-γ could be labeled by periodate-oxidized [α-32P] GTP (GTPoxi). In contrast to GTPoxi labeling of CD3-ξ, (Peter, M. E., Hall, C, Ruhlmann, A., Sancho, J. and Terhorst, C, EMBOJ. 1992. 11: 933), GTP-specific labeling of CD3-γ reached a maximum when nucleotides were added 60 min prior to the cross-linking reaction. As CD3-γ did not contain a known consensus sequence for nucleotide binding and since labeling kinetics of CD3-γ coincided with those of cytosolic GTP-binding proteins, labeling may have been caused by a GTP-binding protein. This putative protein was not T cell specific because labeling of CD3-γ could also be achieved when expressed in the endoplasmic reticulum of Chinese hamster ovary (CHO) cells. In CHO cells, labeling by GTPoxi took place only when CD3-γ was associated with CD3-ξ, whereas labeling could not be established upon association of CD3-γ with CD3-δ or TcR α. The observation that CD3-γ was labeled without leaving the endoplasmic reticulum led to the hypothesis that the association of CD3-γ with a GTP-binding protein might be involved in an early step of the TcR/CD3 complex formation or transport.