The multichain T cell antigen receptor (TCR) is composed of an antigen binding (alpha/beta) domain and associated signal-transducing complexes, the CD3 (gamma, delta, and epsilon) and the zeta chains. The zeta chain (TCR zeta) plays a key role in signal transduction. We show here that TCR ligation induces association of tyrosine-phosphorylated TCR zeta with the detergent-insoluble cell fraction. The microfilament poison, cytochalasin D, disrupts this association and enhances the coprecipitation of actin with TCR zeta after receptor ligation. This microfilament association is specific to TCR-associated polypeptides containing at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Mapping studies using transfectants and chimeric TCR zeta chain constructs suggest that the third ITAM is necessary and sufficient for association, if the distal tyrosine is intact. This cytoskeletal association is directly correlated with IL-2 production, and ligation of TCR on immature thymocytes does not induce TCR zeta-cytoskeleton association. These data thus provide direct evidence of a developmentally regulated activation-dependent interaction between a lymphocyte antigen receptor and the actin cytoskeleton.

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Abstract. To study the molecular basis for organized pigment granule transport, procedures were developed to lyse melanophores of Tilapia mossambica under conditions in which pigment granule movements could be reactivated. Gentle lysis of the melanophores resulted in a permeabilized cell model, which, in the absence of exogenous ATP, could undergo multiple rounds of pigment granule aggregation and dispersion when sequentially challenged with epinephrine and cAMP. Both directions of transport required ATP, since aggregation or dispersion in melanophores depleted of nucleotides could be reactivated only upon addition of MgATP or MgATP plus cAMP, respectively. Differences between the nucleotide sensitivities for aggregation and dispersion were demonstrated by observations that aggregation had a lower apparent Km for ATP than did dispersion and could be initiated at a lower ATP concentration. Moreover, aggregation could be initiated by ADP, but only dispersion could be reactivated by the thiophosphate ATP analog, ATPTS. The direction of pigment transport was determined solely by cAMP, since pigment granules undergoing dispersion reaggregated when cAMP was removed, and those undergoing aggregation dispersed when cAMP was added. These results provide evidence that pigment granule motility may be based on two distinct mechanisms that are differentially activated and regulated to produce bidirectional movements.

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The zeta subunit of the TCR complex targets receptor surface expression, is phosphorylated on tyrosine residues upon T cell activation, and is implicated in signal transduction after TCR ligation. Here we show that, although intrathymic expression of the murine TCR-associated zeta-chain relative to the other chains of the Ag receptor complex remains unchanged during early thymocyte development, there is a doubling of TCR-associated zeta-chain surface expression upon thymocyte maturation. The ratio of tyrosine-phosphorylated relative to nonphosphorylated TCR-associated zeta-chain also changes with thymocyte development. This ratio was quantified after the purification and detergent extraction of receptor complexes from freshly isolated immature or mature thymocytes. Immunoprecipitation of the zeta-chain released from the complex allowed for the isolation of the tyrosine-phosphorylated and nonphosphorylated forms of TCR-associated zeta-chain. Intracellular free zeta-chain was characterized by immunoprecipitation after clearing the cell lysate of intact TCR complexes. Densitometric analysis of immunoblots indicated that surface phosphorylated zeta-chain is more abundant in immature relative to mature T cell populations, whereas the inverse is true of intracellular phosphorylated zeta-chain. Surface phosphorylated zeta-chain also migrated at a higher m.w. than its cytoplasmic counterpart, suggesting that it is more highly modified on some or all of its available tyrosines. These findings demonstrate that the stoichiometry and post-translational modification of the TCR complex are regulated, in vivo, and may determine the functional maturation of T cell signaling, selection, and activation.