Flow cytometric resonance energy transfer measurements support the association of a 95-kDa peptide termed T27 with the 55-kDa Tac peptide.

Abstract

Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes of a 95-kDa peptide. The T27 antigen is widely distributed, being expressed on B lymphocytes, monocytes, and adult Tleukemic cells but not on polymorphonuclear leukocytes or platelets. There was a low level of T27 expression on resting T cells that increased on T-cell activation. In preliminary studies, the OKT27b antibody coprecipitated a 55-kDa peptide, as well as the 95-kDa peptide, from the radiolabeled cells of the HuT 102B2 cell line. Preclearance with anti-Tac, a monoclonal antibody to the 55-kDa peptide of the multichain interleukin 2 receptor, removed the 55-kDa but not the 95-kDa peptide from subsequent OKT27b immunoprecipitates of HuT 102B2 extracts, suggesting the possibility that the T27 peptide was associated with the Tac peptide. However, the precipitation of the p55 Tac peptide by OKT27b was quite inconsistent. Thus, additional information was sought using a flow cytometric energy transfer technique to provide a physical estimation of the proximity between the Tac and the T27 peptides. The flow cytometric version of the fluorescence resonance energy transfer technique permits the determination of inter-and intramolecular distances at 2-to 10-nm levels on a cell-by-cell basis. Using this approach, there was a mean energy transfer of 7.3% with HuT 102B2 cells when fluorescein isothiocyanate anti-Tac served as the donor and tetramethylrhodamine isothiocyanate OKT27 served as the acceptor. In contrast, there was no energy transfer in comparable studies observed when fluorescein anti-Tac and rhodamine anti-transferrin receptor antibodies were used. These observations support the conclusion that there is a close nonrandom proximity in HuT 102B2 cells between the 95-kDa peptide identified by the OKT27 monoclonal antibody and the p55 Tac peptide of the multichain interleukin 2 receptor.T lymphocytes stimulated with antigen or mitogen produce interleukin 2 (IL-2) (1, 2). T-cell growth is dependent on the interaction of IL-2 with high-affinity IL-2 receptors that are not present on resting cells but are induced and expressed on T cells after activation (3-5). There are at least two classes of IL-2 receptors that differ markedly in their affinities for IL-2 (6). Both classes of receptors share the same Tac peptide (55 kDa) (4,5). To define the molecular basis for high-and low-affinity receptors and to determine the mechanism whereby IL-2 communicates a signal to the nucleus, we have investigated the possibility that the IL-2 receptor is a complex with multiple peptide chains in addition to the one identified by anti-Tac (7). Using crosslinking methodology, we have identified a non-Tac IL-2 binding peptide of 75 kDa and have proposed that the 75-kDa IL-2 binding peptide is associated with the 55-kDa Tac peptide to form the highaffinity IL-2 receptor complex (7). Recently, two additional monoclonal antibodies, OKT27 and OKT27b, were developed in an effort to identify distinct human T-cell activation antig...

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“…The average E values, given in percentages for the specified receptor pairs, are shown in the left columns of Table 1 (untreated). E values above 5% indicate an average molecular proximity of less than 10 nm between the fluorescent dyes in the circumstances of our measurements (34,35,38,(42)(43)(44)(45)(46). We observed transfer efficiencies above 5% in the cases of Pgp/CD44, Pgp/CD59, and Pgp/CTX ( Table 1).…”

“…The energy transfer efficiency (E) values between xFITC-and xTRITC-conjugated Fab fragments bound to the cell surface were calculated on a cell-by-cell basis and displayed as energy transfer frequency distribution histograms with the specialized software (39 -41). Based on previous experience (34,35,38,(42)(43)(44)(45)(46), E values above 5% are considered indicative of molecular proximity within the range of 10 nm. The calculated average molecular proximity is also sensitive to the relative membrane density of the two labeled proteins (A/D ratio), and only the donor-proximal acceptors are able to quench donor excitation with this type of energy transfer.…”

“…FRET data were measured on a cell-by-cell basis by using the FCET method previously developed in our laboratory (34,35,38,(42)(43)(44)(45)(46). Using this approach, the molecular proximity of the cell surface proteins, labeled by monoclonal antibodies tagged with donor and acceptor fluorescent probes, was estimated based on the E values measured.…”

The IL-2/IL-2 Receptor System. Involvement of a Novel Receptor Subunit, .GAMMA. Chain, in Growth Signal Transduction.

“…Others have also found molecules associated with IL-2R, which included ICAM-I (p95) (Szollosi et al 1987), HLA class I (p40-42) (Sharon et al 1988), p22 and p40 (Saragovi and Malek 1990), p95-110 (Colamonici et al 1990) and p100 (Sharon et al 1990), but there is no evidence that these molecules contribute to the formation of functional IL-2R complex. In our experimental system with no chemical crosslinker, only the p64 molecule was detected in the complexes of both functional high-affinity IL-2R and functional intermediate-affinity IL-2R.…”