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

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 (GTP_oxi). In contrast to GTP_oxi 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 GTP_oxi 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.