Homooligomerization of the Cytoplasmic Domain of the T Cell Receptor ζ Chain and of Other Proteins Containing the Immunoreceptor Tyrosine-Based Activation Motif

Abstract

Antigen receptors on T cells, B cells, mast cells, and basophils all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. How clustering of receptor extracellular domains leads to phosphorylation of cytoplasmic domain ITAMs is not known, and little structural or biochemical information is available for the ITAM-containing cytoplasmic domains. Here we investigate the conformation and oligomeric state of several immune receptor cytoplasmic domains, using purified recombinant proteins and a variety of biophysical and biochemical techniques. We show that all of the cytoplasmic domains of ITAM-containing signaling subunits studied are oligomeric in solution, namely, T cell antigen receptor zeta, CD3epsilon, CD3delta, and CD3gamma, B cell antigen receptor Igalpha and Igbeta, and Fc receptor FcepsilonRIgamma. For zeta(cyt), the oligomerization behavior is best described by a two-step monomer-dimer-tetramer fast dynamic equilibrium with dissociation constants in the order of approximately 10 microM (monomer-dimer) and approximately 1 mM (dimer-tetramer). In contrast to the other ITAM-containing proteins, Igalpha(cyt) forms stable dimers and tetramers even below 10 microM. Circular dichroic analysis reveals the lack of stable ordered structure of the cytoplasmic domains studied, and oligomerization does not change the random-coil-like conformation observed. The random-coil nature of zeta(cyt) was also confirmed by heteronuclear NMR. Phosphorylation of zeta(cyt) and FcepsilonRIgamma(cyt) does not significantly alter their oligomerization behavior. The implications of these results for transmembrane signaling and cellular activation by immune receptors are discussed.

Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form

“…Considering the unstructured nature of ζ cyt , the question as to whether the protein remains disordered or undergoes a structural transition to a folded form in the ζ cyt dimer is of fundamental importance. According to our previously reported circular dichroic data [7], specific dimerization of ζ cyt does not induce detectable secondary or tertiary structure. Because of the high sensitivity of NMR chemical shifts to structural changes at atomic resolution not detected with optical methods, NMR spectroscopy is unparalleled in its ability to provide detailed structural information on unfolded proteins [9].…”

“…Signaling through the ζ chain is mediated by a triplicate of conserved cytoplasmic sequence motifs, termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), where tyrosine residues are phosphorylated upon TCR engagement in an early and obligatory event in the signaling cascade. We and others have previously reported that the 13-kDa ζ cyt oplasmic domain (ζ cyt ) is intrinsically disordered [6][7][8]. Recently, we have found that this protein shows clear signs of specific dimerization [7].…”

“…We and others have previously reported that the 13-kDa ζ cyt oplasmic domain (ζ cyt ) is intrinsically disordered [6][7][8]. Recently, we have found that this protein shows clear signs of specific dimerization [7]. Considering the unstructured nature of ζ cyt , the question as to whether the protein remains disordered or undergoes a structural transition to a folded form in the ζ cyt dimer is of fundamental importance.…”

Cells diversify transmembrane signaling through the controlled chaos of protein disorder

“…Surprisingly, a striking difference between the two families is observed in structural patterns of intracellular signal-generating regions. Recently, using a variety of biophysical techniques 1,4,9 and prediction algorithms, 3,5 we found that while signaling by SRs is mediated through intracellular well-defined protein structures (Fig. 1) [10][11] (4) The modular assembly of MIRRs (Fig.…”

“…Recent findings show that while ligand binding outside the cell is mediated through well-structured protein domains, intracellular signaling domains of many receptors represent intrinsically disordered regions (IDRs), the regions that lack a well-defined three-dimensional structure under physiological conditions. [1][2][3][4][5] Intriguingly, protein intrinsic disorder is a characteristic feature of the cytoplasmic signaling domains of those receptors, in which recognition and signaling are mediated by separate protein chains. 1,4,5 Our studies of these signaling-related IDRs reveal several unusual and previously unreported biophysical phenomena 1,4,[6][7][8][9] that not only facilitate a rethinking process of the fundamental paradigms in protein biophysics but also open new perspectives on the molecular mechanisms of receptor signaling with multiple applications in biology and medicine.…”