Antigen-receptor induced clonal expansion and deletion of lymphocytes are impaired in mice lacking HS1 protein, a substrate of the antigen-receptor-coupled tyrosine kinases.

HS1, an intracellular protein expressed specifically in hematopoietic cells, is rapidly tyrosine phosphorylated after cross-linking of antigen receptors on B and T lymphocytes, implicating involvement of this molecule in the signal transduction pathways from the antigen receptors as a substrate of membrane-associated tyrosine kinase(s). The development of lymphoid cells in HS1-deficient mice, generated through gene targeting, appeared normal. However, antibody production to T-independent antigen and proliferative responses of splenic B and T cells after cross-linking of the antigen receptors were impaired in these mutant mice. Furthermore, B cells in the peritoneal cavity of the mutant mice were resistant to multivalent cross-linking of the antigen receptor, which causes apoptosis of such cells in normal mice. Crossing the HS1-deficient mice with the mice harboring transgenes encoding alpha and beta chains of T-cell antigen receptor against a male H-Y antigen resulted in a progeny that demonstrated a significantly impaired ability of thymic negative selection. These results indicate that HS1 is a novel molecule involved in the antigen-receptor-derived signaling pathways and plays important roles not only in clonal expansion, but also in clonal deletion of B and T cells.     

Trafficking of varicella-zoster virus glycoprotein gI: T(338)-dependent retention in the trans-Golgi network, secretion, and mannose 6-phosphate-inhibitable uptake of the ectodomain

The trans-Golgi network (TGN) is putatively the site where varicella-zoster virus is enveloped. gE is targeted to the TGN by selective retrieval from the plasmalemma in response to signaling sequences in its endodomain. gI lacks these sequences but forms a complex with gE. We now find that gI is targeted to the TGN and plasma membrane when expressed in Cos-7 cells; nevertheless, surface labeling revealed that gI is not retrieved from the plasma membrane. TGN targeting of gI depended on the T(338) of its endodomain and was lost when T(338) was deleted or mutated to A, S, or D. The endodomain of gI was sufficient, if it contained T(338), to target a fusion protein containing the ectodomain of the human interleukin-2 receptor to the TGN. A truncated protein consisting only of the gI ectodomain was secreted and taken up by nontransfected cells. This uptake of the secreted gI ectodomain was blocked by mannose 6-phosphate. Following cotransfection, both gI and gE were retrieved to the TGN from the plasma membrane in 26.7% of cells, neither gI nor gE was internalized in 18.3%, and gE was retrieved to the TGN while gI remained at the plasma membrane in 55%. We suggest that the T(338) of its endodomain is necessary to retain gI in the TGN; moreover, because gI and gE interact, the signaling sequences of each glycoprotein reinforce one another in ensuring that both glycoproteins are concentrated in the TGN yet remain on the cell surface.     

Transmembrane helix of novel oncogene with kinase-domain (NOK) influences its oligomerization and limits the activation of RAS/MAPK signaling

Ligand-dependent or independent oligomerization of receptor protein tyrosine kinase (RPTK) is often an essential step for receptor activation and intracellular signaling. The novel oncogene with kinase-domain (NOK) is a unique RPTK that almost completely lacks an ectodomain, expresses intracellularly and activates constitutively. However, it is unknown whether NOK can form oligomer or what function oligomerization would have. In this study, two NOK deletion mutants were generated by either removing the ectodomain (NOKΔECD) or including the endodomain (NOK-ICD). Co-immunoprecipitation demonstrated that the transmembrane (TM) domain of NOK was essential for its intermolecular interaction. The results further showed that NOK aggregated more closely as lower order oligomers (the dimer- and trimer-sized) than either deletion mutant did since NOK could be cross-linked by both Sulfo-EGS and formaldehyde, whereas either deletion mutant was only sensitive to Sulfo-EGS. Removing the NOK TM domain (NOK-ICD) not only markedly promoted higher order oligomerization, but also altered the subcellular localization of NOK and dramatically elevated the NOK-mediated constitutive activation of extracellular signal-regulated kinase (ERK). Moreover, NOK-ICD but not NOK or NOKΔECD was co-localized with the upstream signaling molecule RAS on cell membrane. Thus, TM-mediated intermolecular contacting may be mainly responsible for the constitutive activation of NOK and contribute to the autoinhibitory effect on RAS/MAPK signaling.     

CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.     

Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain

The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.     

Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.     

Current Understanding of Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) Signaling in T-Cell Biology and Disease Therapy

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an immune checkpoint molecule that is mainly expressed on activated T cells and regulatory T (Treg) cells that inhibits T-cell activation and regulates immune homeostasis. Due to the crucial functions of CTLA-4 in T-cell biology, CTLA-4-targeted immunotherapies have been developed for autoimmune disease as well as cancers. CTLA-4 is known to compete with CD28 to interact with B7, but some studies have revealed that its downstream signaling is independent of its ligand interaction. As a signaling domain of CTLA-4, the tyrosine motif plays a role in inhibiting T-cell activation. Recently, the lysine motif has been shown to be required for the function of Treg cells, emphasizing the importance of CTLA-4 signaling. In this review, we summarize the current understanding of CTLA-4 biology and molecular signaling events and discuss strategies to target CTLA-4 signaling for immune modulation and disease therapy.     

Activation of c-jun N-terminal kinase by 4-1BB (CD137), a T cell co-stimulatory molecule

It has been widely accepted that T cell activation requires two signals; one from the binding of the antigen/major histocompatibility complex to the T-cell receptor (TCR)/CD3 complex and the other from the interaction between a surface molecule on antigen presenting cells and its receptor on T cells. The second signal is considered as co-stimulatory and the B7/CD28 pair has been well studied as a prototype. Recently 4-1BB (CD137) has been characterized as another co-stimulatory molecule for T cell activation. However, unlike the CD28/B7 pair, 4-1BB and its ligand 4-1BBL constitute a member of the tumor necrosis factor (TNF) receptor/TNF pair superfamily. The signaling mechanism of 4-1BB has not been revealed in detail. To investigate whether 4-1BB takes the signaling pathways analogous to those for TNF receptors, we generated polyclonal antibodies against human 4-1BB and 4-1BBL and established stable transfectants of the receptor and the ligand with a high level of cell surface expression. Over-expression of h4-1BB was found to result in the activation of c-Jun N-terminal kinase (JNK) in the human embryonic kidney cell line 293. In T cells, it has been previously demonstrated that JNK activation requires dual signals such as the ligation of TCR/CD3 complex plus CD28 co-stimulation or PMA plus ionomycin. The JNK activation by 4-1BB in Jurkat T cells was also found to require stimulation of the TCR/CD3 complex, consistent with the notion that 4-1BB functions as a co-stimulatory molecule for T cell activation.     

The evolution of vertebrate antigen receptors: a phylogenetic approach

Classical T cells, those with alpha beta T-cell receptors (TCRs), are an important component of the dominant paradigm for self-nonself immune recognition in vertebrates. alpha beta T cells recognize foreign peptide antigens when they are bound to MHC molecules on the surfaces of antigen-presenting cells. gamma delta T cells bear a similar receptor, and it is often assumed that these T cells also require specialized antigen-presenting molecules for immune recognition, which we term "indirect antigen recognition." B-cell receptors, or immunoglobulins, bind directly to antigens without the help of a specialized antigen-presenting molecule. Phylogenetically, it has been assumed that T-cell receptors and the genes that encode them are a monophyletic group, and that "indirect" antigen recognition evolved before the split into two types of TCR. Recently, however, it has been proposed that gamma delta-TCRs bind directly to antigens, as do immunoglobulins (Ig's). This calls into question the null hypothesis that indirect antigen recognition is a common characteristic of TCRs and, by extension, the hypothesis that all TCR gene sequences form a monophyletic group. To determine whether alternative explanations for antigen recognition and other historical relationships among TCR genes might be possible, we performed phylogenetic analyses on amino acid sequences of the constant and variable regions which encode the basic subunits of TCR and Ig molecules. We used both maximum-parsimony and genetic distance-based methods and could find no strong support for the hypothesis of TCR monophyly. Analyses of the constant region suggest that TCR gamma or delta sequences are the most ancient, implying that the ancestral immune cell was like a modern gamma delta T cell. From this gamma delta-like ancestor arose alpha beta T cells and B cells, implying that indirect antigen recognition is indeed a derived property of alpha beta-TCRs. Analyses of the variable regions are complicated by strong selection on antigen-binding sequences, but imply that direct antigen binding is the ancestral condition.     

Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor

MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glycoforms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3zeta endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-gamma and IL-17), and elicit brisk killing of MUC1(+) tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells.     

Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.

Artificial receptors provide a promising approach to target T lymphocytes to tumor antigens. However, the receptors described thus far produce either an activation or a co-stimulatory signal alone, thus limiting the spectrum of functions accomplished by the genetically modified cells. Here we show that human primary T lymphocytes expressing fusion receptors directed to prostate-specific membrane antigen (PSMA) and containing combined T-cell receptor-zeta (TCRzeta), and CD28 signaling elements, effectively lyse tumor cells expressing PSMA. When stimulated by cell-surface PSMA, retrovirally transduced lymphocytes undergo robust proliferation, expanding by more than 2 logs in three weeks, and produce large amounts of interleukin-2 (IL-2). Importantly, the amplified cell populations retain their antigen-specific cytolytic activity. These data demonstrate that fusion receptors containing both TCR and CD28 signaling moieties are potent molecules able to redirect and amplify human T-cell responses. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of tumor cells that fail to express major histocompatibility complex antigens and co-stimulatory molecules.     

Regulated proteolytic processing of Tie1 modulates ligand responsiveness of the receptor-tyrosine kinase Tie2

Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor-tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane-tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. After Tie1 ectodomain cleavage the newly formed 45-kDa endodomain undergoes additional proteolytic processing mediated by gamma-secretase to generate an amino-terminal-truncated 42-kDa fragment that is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing, we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of cartilage oligomeric protein angiopoietin 1. A key function of gamma-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases cartilage oligomeric protein angiopoietin 1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand after shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggest that regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2.     

人类免疫缺陷病毒1型gp41结构与功能的研究进展

人类免疫缺陷病毒1型(HIV-1)通过其包膜糖蛋白(Env)介导侵入靶细胞.Env由受体特异性结合单位gp120和膜融合单位gp41组成.HIV-1的gp41分为3个功能区:膜外区、跨膜区和膜内区.膜外区是病毒感染时膜融合的主要结构基础;跨膜区通过疏水残基使Env锚定在脂质膜上;膜内区则表现多重功能,参与病毒的感染、复...     

Expression and function of the insulin receptor substrate proteins in cancer

The Insulin Receptor Substrate (IRS) proteins are cytoplasmic adaptor proteins that function as essential signaling intermediates downstream of activated cell surface receptors, many of which have been implicated in cancer. The IRS proteins do not contain any intrinsic kinase activity, but rather serve as scaffolds to organize signaling complexes and initiate intracellular signaling pathways. As common intermediates of multiple receptors that can influence tumor progression, the IRS proteins are positioned to play a pivotal role in regulating the response of tumor cells to many different microenvironmental stimuli. Limited studies on IRS expression in human tumors and studies on IRS function in human tumor cell lines and in mouse models have provided clues to the potential function of these adaptor proteins in human cancer. A general theme arises from these studies; IRS-1 and IRS-4 are most often associated with tumor growth and proliferation and IRS-2 is most often associated with tumor motility and invasion. In this review, we discuss the mechanisms by which IRS expression and function are regulated and how the IRS proteins contribute to tumor initiation and progression.     

Human Vdelta1 gamma-delta T cells exert potent specific cytotoxicity against primary multiple myeloma cells

Abstract Background aims. Human gamma-delta (γδ) T cells are potent effector lymphocytes of innate immunity involved in anti-tumor immune surveillance. However, the Vδ1 γδ T-cell subset targeting multiple myeloma (MM) has not previously been investigated. Methods. Vδ1 T cells were purified from peripheral blood mononuclear cells of healthy donors and patients with MM by immunomagnetic sorting and expanded with phytohemagglutinin (PHA) together with interleukin (IL)-2 in the presence of allogeneic feeders. Vδ1 T cells were phenotyped by flow cytometry and used in a 4-h flow cytometric cytotoxicity assay. Cytokine release and blocking studies were performed. Primary myeloma cells were purified from MM patients' bone marrow aspirates. Results. Vδ1 T cells expanded from healthy donors displayed prominent cytotoxicity by specific lysis against patients' CD38 (+) CD138 (+) bone marrow-derived plasma cells. Vδ1 T cells isolated from MM patients showed equally significant killing of myeloma cells as Vδ1 T cells from normal donors. Vδ1 T cells showed similarly potent cytotoxicity against myeloma cell lines U266 and RPMI8226 and plasma cell leukemia ARH77 in a dose-dependent manner. The interferon (IFN)-γ secretion and Vδ1 T-cell cytotoxicity against myeloma cells was mediated in part through the T-cell receptor (TCR) in addition to involvement of Natural killer-G2D molecule (NKG2D), DNAX accessory molecule-1 (DNAM-1), intracellular cell adhesion molecule (ICAM)-1, CD3 and CD2 receptors. In addition, Vδ1 T cells were shown to exert anti-myeloma activity equal to that of Vδ2 T cells. Conclusions. We have shown for the first time that Vδ1 T cells are highly myeloma-reactive and have therefore established Vδ1 γδ T cells as a potential candidate for a novel tumor immunotherapy.     

Diversity in immune-cell interactions: states and functions of the immunological synapse

The contact-dependent exchange of signals between epithelial and neuronal cells results from close membrane-membrane appositions, which are stabilized for years by polarized adhesion, cytoskeletal assemblies and extracellular scaffold proteins. By contrast, owing to a lack of scaffold proteins, interactions between immune cells such as T lymphocytes and antigen-presenting cells (APCs) comprise a spectrum of structurally diverse and short-lived interaction modes that last from minutes to hours. Signals exchanged between T cells and APCs are generated in a specific contact region, termed the "immunological synapse", that coordinates cytoskeletal dynamics with the T-cell receptor (TCR), the engagement of accessory receptors and membrane-proximal signaling. Recent data shed light on the different physical and molecular interaction modes that occur between T cells and APCs, including their dynamics and transition stages, and their consequences for signaling, activation and T-cell effector function.     

Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy

Background aimsCytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in pre-clinical models, and this efficacy has translated to success in several clinical trials. Many early trials were disappointing in large part because of the lack of proliferation and subsequent persistence of transferred cells. Recent investigations have pointed to the importance of delivering highly proliferative cells, whether of naive or early memory phenotypes.MethodsWe investigated the influence of two common cell culturing methods used in early trials and their relationship to T-cell phenotype and pre-clinical efficacy.ResultsWe observed that stimulation with soluble anti-CD3 antibody OKT-3 and high-dose interleukin-2 produces more effector memory-type T cells with shorter average telomeres when compared with cells generated with the use of CD3/CD28 beads. When used in xenograft models of leukemia, bead-stimulated cells proliferated earlier and to a higher degree than those generated with the use of OKT-3/IL2 and resulted in better disease control despite no difference in distribution or migration throughout the mouse. Inclusion of the known successful clinical 4-1BB endodomain in the CAR could not rescue the function of OKT-3/IL-2–cultured cells. T cells isolated from animals that survived long-term (>120 days) retained a central memory–like phenotype and demonstrated a memory response to a large re-challenge of CD19-positive leukemia.ConclusionsIn summary, we confirm that cells with a younger phenotype or higher proliferative capacity perform better in pre-clinical models and that cell culturing influences cell phenotype seemingly independent of the 4-1BB endodomain in the CAR structure.     

Redirecting mouse CTL against colon carcinoma: superior signaling efficacy of single-chain variable domain chimeras containing TCR-zeta vs Fc epsilon RI-gamma

The structurally related TCR-zeta and Fc receptor for IgE (Fc epsilon RI)-gamma are critical signaling components of the TCR and Fc epsilon RI, respectively. Although chimeric Ab receptors containing zeta and gamma signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the zeta and gamma subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-zeta and scFv-gamma receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-zeta chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-gamma receptors. T cells expressing scFv-zeta chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the zeta signaling pathway.     

Structural Studies of T200 Glycoprotein and the Il-2 Receptor

Abstract

Immunological analysis of the cell surface of hematopoietic cells has led to the identification of many different cell membrane molecules, some of which have well-defined functions as receptors. In general, however, the role of most lymphocyte cell surface molecules remains ill-defined even in cases in which antibody inhibition studies have given some insight into the biological processes in which they participate. Here we describe molecular and biochemical studies of T200 glycoprotein (leukocyte-common antigen) and the IL-2 receptor which illustrate the kinds of approaches that can be currently used to characterize individual molecules. T200 glycoprotein is a large M_r glycoprotein found exclusively on leukocytes. However, the exact M_r varies in a cell-type-specific fashion and this property is conserved between different species. Comparison of the rat, mouse and human cDNA sequences show that the large cytoplasmic portion of the molecule is well-conserved, approximately 90%, whereas the exterior portion is only about 50% homologous. Cell-type-specific differences in the primary sequence of the molecule have been identified in the N-terminal portion of the molecules. In contrast to T200, the function of the IL-2 receptor is well-known. The interaction of IL-2 with its receptor provides a growth signal that determines the magnitude and duration of T-cell responses. Limited proteolysis studies provide the first direct biochemical evidence that the external region of the IL-2 receptor consists of two independent domains. ¹²⁵I-labeled IL-2 has been chemically crosslinked to the receptor and proteolytic cleavage of the crosslinked product indicates that IL-2 is selectively bound to the N-terminal domain of the receptor.