Disulfide-bonded Homodimers Research Articles

P0968THE INFLUENCE OF ALPHA KLOTHO ON NPP1 ACTIVITY IN CULTURED RAT PODOCYTES

Abstract Background and Aims Kidney is the principal contributor to circulating αKlotho (αKL), and simultaneously, constitutes the major organ where circulating αKlotho is taken up from the circulation. Klotho is mainly expressed in proximal renal tubule and in distal tubular epithelium. In the kidney soluble αKlotho acts from the urinary luminal side as an autocrine or paracrine enzyme to regulate transporters and ion channels. Recently, it has been demonstrated that the glomerulus is another source of αKlotho production. NPP1 is the best characterized nucleotide pyrophosphatases/phosphodiesterases (NPP) family member, encoded by the ENPP1 gene, and is a type II transmembrane glycoprotein that forms disulfide-bonded homodimers in the plasma membrane and in the mineral-depositing matrix vesicles. The major function of NPP1 is generation of PPi from adenosine triphosphate (ATP). Recently, we demonstrated that extracellular nucleotides stimulate an increase in glomerular albumin permeability, in part due to glomerular NO production and actin reorganization in podocytes . Furthermore, both αKlotho and NPP1 regulate insulin-dependent signaling. Thus, a key aim of this study is to fully examine the role of αKlotho on NPP1 function and its role in the protection of glomerular filtration barrier and podocyte function in diabetes. Method We measured glomerular permeability to albumin (Palb) in a single isolated rat glomerulus based on the video-microscopy method and permeability to albumin across the podocyte monolayer. Expression of αKlotho and NPP1 was confirmed in podocytes using Western blotting and immunofluorescence. Ecto-nucleotide pyrophosphatase/phosphodiesterase activity in podocytes was assessed using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. The ATP concentration was measured using luciferin-luciferase reaction. Results Since NPP1 and Klotho regulate insulin-dependent signaling we investigated the role of Klotho on NPP1 dimerization and NPPs activity. Podocyte treatment withαKlotho (0.5 nM, 30 min) in non-reducing conditions (maleimide, 100 mM) increased the disulfide bond formation of NPP1 (Fig. 1A). The percentage of dimerized (260 kDa) NPP1 increased approximately by 45%. This effect was abolished in the presence of ATP (100 µM, 30 min). As a positive control, we used hydrogen peroxide (100 μM, 5 min) which induces oxidative disulfide bond formation (Fig. 1A). We also demonstrated time-dependent decrease of NPPs activity in the presence of αKL (Fig. 1B). Klotho also induces an increase of extracellular ATP concentration by 76% (from 3.19±0.01 to 5.62±0.43, n=6, P<0.05). Moreover, we confirmed the role of soluble αKlotho in ATP-evoked increases of glomerular and podocyte albumin permeability. Conclusion Summarizing, we have clearly demonstrated an interaction between αKL and NPP1 in podocytes. Moreover, we observed the protective role of αKlotho in podocyte permeability to albumin.

The β2-Microglobulin–Free Heterodimerization of Rhesus Monkey MHC Class I A with Its Normally Spliced Variant Reduces the Ubiquitin-Dependent Degradation of MHC Class I A

The MHC class I (MHC I) molecules play a pivotal role in the regulation of immune responses by presenting antigenic peptides to CTLs and by regulating cytolytic activities of NK cells. In this article, we show that MHC I A in rhesus macaques can be alternatively spliced, generating a novel MHC I A isoform (termed "MHC I A-sv1") devoid of α(3) domain. Despite the absence of β2-microglobulin (β2m), the MHC I A-sv1 proteins reached the cell surface of K562-transfected cells as endoglycosidase H-sensitive glycoproteins that could form disulfide-bonded homodimers. Cycloheximide-based protein chase experiments showed that the MHC I A-sv1 proteins were more stable than the full-length MHC I A in transiently or stably transfected cell lines. Of particular interest, our studies demonstrated that MHC I A-sv1 could form β2m-free heterodimers with its full-length protein in mammalian cells. The formation of heterodimers was accompanied by a reduction in full-length MHC I A ubiquitination and consequent stabilization of the protein. Taken together, these results demonstrated that MHC I A-sv1 and MHC I A can form a novel heterodimeric complex as a result of the displacement of β2m and illustrated the relevance of regulated MHC I A protein degradation in the β2m-free heterodimerization-dependent control, which may have some implications for the MHC I A splice variant in the fine tuning of classical MHC I A/TCR and MHC I A/killer cell Ig-like receptor interactions.

The inability of vaccinia virus A33R protein to form intermolecular disulfide-bonded homodimers does not affect the production of infectious extracellular virus

The orthopoxvirus protein A33 forms a disulfide-bonded high molecular weight species that could be either a homodimer or a heteromultimer. The protein is a major target for neutralizing antibodies and the majority of antibodies raised against A33 only recognize the disulfide-bonded form. Here, we report that A33 is present as a disulfide-bonded homodimer during infection. Additionally, we examined the function of intermolecular disulfide bonding in A33 homodimerization during infection. We show that the cysteine at amino acid 62 is required for intermolecular disulfide bonding, but not dimerization as this mutant was still able to form homodimers. To investigate the role of disulfide-bonded homodimers during viral morphogenesis, recombinant viruses that express an A33R with cysteine 62 mutated to serine were generated. The recombinant viruses had growth characteristics similar to their parental viruses, indicating that intermolecular disulfide-bonded homodimerization of A33 is not required for its function.

Penumbra encodes a novel tetraspanin that is highly expressed in erythroid progenitors and promotes effective erythropoiesis

AbstractIn a search for new genes involved in the regulation of erythropoiesis, we identified murine Penumbra cDNA from a multipotent hematopoietic cell line based on its predominant expression in erythroblasts. Subsequently, we identified the human PENUMBRA from a bone marrow cDNA library. Penumbra is a new member of the tetraspanin superfamily of membrane proteins, many of which are thought to function as organizers of supramolecular signaling complexes. Human and murine Penumbras contain 283 amino acids and are 97% identical. The human PENUMBRA gene is mapped to chromosome 7q32, a hot spot for deletions in myelodysplastic syndromes and acute myelogenous leukemias. Penumbra is targeted to the cell surface and forms disulfide-bonded homodimers. To study the effects of Penumbra deletions, we created a knockout mouse model by gene targeting. Penumbra−/− mice develop massive splenomegaly, basophilic macrocytic red blood cells, and anemia as they age. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/− mouse. EMX exhibits ineffective erythropoiesis in the presence of erythropoietin, a defect that is reversed by reexpression of Penumbra. These findings indicate that Penumbra has a positive function in erythropoiesis and its deletion or mutation may result in anemia.

Inactivation of active and latent transforming growth factor beta by free thiols: Potential redox regulation of biological action

Transforming growth factor-β (TGF-β) is a multifunctional cytokine with important roles in inflammation, wound repair, and cancer. Cells secrete TGF-β as a latent protein complex, consisting of disulfide-bonded homodimers of growth factor and latency-associated propeptide. Latency regulates extracellular TGF-β action by controling the levels of active growth factor available. We report here that active and latent TGF-β were inactivated in vitro by reduction of the growth factor dimer under physiological conditions. We also demonstrate that the latency-associated propeptide has chaperone-like activity and partially protects TGF-β from inactivation.TGF-β inactivation occured upon incubation with the physiological redox agents, cysteine, homocysteine, and reduced glutathione. Inactivation was temperature- and dose-dependent. While inactivation by physiological concentrations of redox agents was partial at 37°C, active and latent TGF-β were completely inactivated by raising the temperature in the presence of the redox agents. The mechanism of TGF-β inactivation involved the generation of biologically inactive growth factor monomer and required the presence of free thiol groups, since thiol blockers protected TGF-β from reduction.We conclude that non-enzymatic redox reactions may be involved in the regulation of extracellular TGF-β activity. This might be of particular relevance in wound repair (e.g. in burns), as a mechanism protecting from excess TGF-β activity, as well as in conditions involving redox dysregulation, such as reperfusion injury of the heart, Alzheimer's disease, and cancer.

Ly-49 Receptors and Their Functions

Ly-49 receptors are lectin-like type II transmembrane disulfide-bonded homodimers expressed on natural killer (NK) cells and some T-cell subsets. Cell-mediated cytotoxicity and release of cytokines/chemokines are functions regulated by Ly-49 recognition of class I major histocompatibility complex proteins (MHC-I) or virus-encoded MHC-like product(s). Here we examine diversity and conservation found within the Ly-49 gene family and explore the importance of polymorphism in Ly-49 receptor expression, specificity, and function. Several parallels are evident between Ly-49 receptors in rodents and killer Ig-related (KIR) receptors in humans, including receptor gene amplification and diversification, expression patterns, MHC-I specificity, shared signaling, and ultimate effects on NK-cell functions. These similarities suggest that insights gained in defining Ly-49 receptor functions in small animal models could have direct relevance to existing clinical challenges where there may be opportunities to manipulate human NK cells and KIR receptors for therapeutic benefit.

Lymphoblastoid cells express HLA-B27 homodimers both intracellularly and at the cell surface following endosomal recycling.

The MHC class I allele HLA-B27 is very strongly associated with development of autoimmune spondyloarthritis, although the disease mechanism remains unknown. Class I molecules classically associate in the endoplasmic reticulum (ER) with beta2-microglobulin (beta(2)m) and antigenic peptides for cell surface expression and presentation to T cells. We have previously shown that HLA-B27 is capable of forming beta(2)m-free disulfide-bonded homodimers in vitro. Here we show that HLA-B27 forms disulfide-bonded homodimers in vivo by two distinct pathways. HLA-B27 homodimers form in the ER but appear unable to egress to the cell surface in human cells. Cell surface HLA-B27 homodimers are abundantly expressed in a variety of lymphoid cell lines. Experiments with inhibitors indicate that HLA-B27 homodimers can arise from cell-surface heterodimers via an endosome-dependent recycling pathway. HLA-B27 homodimer expression on the cell surface of 721.220 is dependent on the unpaired cysteine(67) and is inhibited by restoration of tapasin function or by incubation with peptides that bind strongly to HLA-B27 heterodimers. Cell surface expressed HLA-B27 homodimers are likely to be immunologically reactive ligands for NK family immunoreceptors and, hence, could play a pathogenic role in spondyloarthritis.

HLA-B27: natural function and pathogenic role in spondyloarthritis

Chapter summaryThe human leukocyte antigen HLA-B27 is strongly associated with development of a group of inflammatory arthritides collectively known as the spondyloarthritides. We have set out to define the natural immunological function of HLA-B27, and then to apply this knowledge to understand its pathogenic role. Human leukocyte antigen class 1 molecules bind antigenic peptides for cell surface presentation to cytotoxic T lymphocytes. HLA-B27 binds and presents peptides from influenza, HIV, Epstein-Barr virus, and other viruses. This leads to vigorous and specific cytotoxic T lymphocyte responses, which play an important role in the body's immune response to these viruses. HLA-B27 thus carries out its natural function highly effectively. Although many theories have been proposed to explain the role of HLA-B27 in the pathogenesis of spondyloarthropathy, we favour those postulating that the pathogenic role of HLA-B27 stems from its natural function. For example, the 'arthritogenic' peptide hypothesis suggests that disease results from the ability of HLA-B27 to bind a unique peptide or a set of antigenic peptides. Additionally, a number of lines of evidence from our laboratory and other laboratories have suggested that HLA-B27 has unusual cell biology. We have recently demonstrated that HLA-B27 is capable of forming disulfide-bonded homodimers. These homodimers are expressed on the cell surface and are ligands for a number of natural killer and related immunoreceptors, expressed on a variety of cell types including natural killer cells, T lymphocytes and B lymphocytes, and members of the monocyte/macrophage lineage. We are currently investigating the possibility that such interactions could be involved in disease pathogenesis.

Real-time Monitoring of the Interactions of Transforming Growth Factor-β (TGF-β) Isoforms with Latency-associated Protein and the Ectodomains of the TGF-β Type II and III Receptors Reveals Different Kinetic Models and Stoichiometries of Binding

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-beta can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-beta isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-beta molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.

A novel type of mutation in the cysteine rich domain of the RET receptor causes ligand independent activation.

Multiple endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome, which involves the triad of MTC, pheochromocytoma, and hyperparathyroidism. Missense mutations in one of six cysteine codons in the extracellular cysteine-rich domain of the RET proto-oncogene predispose to this disease. These mutations cause ligand-independent constitutive activation of the tyrosine kinase receptor by the formation of disulfide-bonded homodimers. We examined a different type of mutation, which results in an additional cysteine in the cysteine rich domain. A duplication of 9 bp in the first case resulted in an insertion of three amino acids between codon 633 and 634. In the second case a 12 bp duplication in exon 11 results in four additional amino acids between codon 634 and 635. Here we demonstrate that an additional cysteine causes a ligand independent dimerization of the RET receptor in transfected NIH3T3 cells, which results in an activation of the intracellular tyrosine kinase.

Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry.

Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural heterogeneity, multiple glycosylation sites, and different forms. Here, a top-down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851, and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduced Fcepsilon(2-3-4)(2) protein yielded a peptide mixture with molecular weight values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc-(hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference.

The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein.

Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.

Impaired Activation and Binding of the Erythropoietin Receptor by a Mutant gp55 of Friend Spleen Focus-Forming Virus, Which Has a Cytoplasmic Domain

A murine erythroleukemogenic retrovirus, Friend spleen focus-forming virus, encodes an envelope protein-like membrane glycoprotein (gp55) in its defectiveenvgene which is responsible for activation of the erythropoietin receptor (EpoR) and the abnormally rapid proliferation of erythroid precursor cells. The S34 mutant gp55, which possesses an additional cytoplasmic domain, is nonpathogenic and its processing to the cell surface is severely reduced compared to that of the wild-type gp55. In this study, we found that the S34 mutant gp55 neither binds to nor activates the EpoR. The S34 mutant gp55 formed disulfide-bonded homodimers in the rough endoplasmic reticulum (RER) membrane much less efficiently than the wild-type gp55, which is consistent with the proposal that homodimer formation is a prerequisite for gp55 to be exported from the RER. We found that the wild-type gp55 that is bound to EpoR in the RER consists of a large number of monomers and a small number of dimers, suggesting that monomers of the S34 mutant gp55 have lost the ability to bind to the EpoR. The 1-bp insertion present in the wild-type gp55 gene, causing a loss of the cytoplasmic domain, is essential for pathogenicity in that it renders the encoded protein capable of both binding to the EpoR and transport to the cell surface.

Full activation of MEN2B mutant RET by an additional MEN2A mutation or by ligand GDNF stimulation

Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).

Refolding of single-chain Fv with C-terminal cysteine (sFv'): Formation of disulfide-bonded homodimers of Anti-<italic>c</italic>-<italic>erb</italic>B-2 and Anti-digoxin sFv'

Refolding of single-chain Fv with C-terminal cysteine (sFv'): Formation of disulfide-bonded homodimers of Anti-<italic>c</italic>-<italic>erb</italic>B-2 and Anti-digoxin sFv'

Selective secretion of alternatively spliced fibronectin variants.

We demonstrate that the alternatively spliced variable (V) region of fibronectin (FN) is required for secretion of FN dimers during biosynthesis. Alternative splicing of the V segment of the rat FN transcript generates three subunit variants (V120, V95, V0) that differ by the inclusion or omission of an additional 120 or 95 amino acids. We are exploring the functions of this segment by expressing variant cDNAs in normal and transformed fibroblasts. Like FN itself, the cDNA-encoded polypeptides (deminectins [DNs]) containing the V120 or V95 segment are efficiently secreted as disulfide-bonded homodimers. However, few homodimers of DNs lacking this region, V0 DNs, are secreted. V0 homodimers do form inside the cell, as demonstrated by biosynthetic analyses of dimer formation and secretion using pulse-chase and time course experiments, but these dimers seldom reach the cell surface and are probably degraded intracellularly. Coexpression of V0 and V120 subunits results in intracellular formation of three types of dimers, V0-V0, V0-V120, and V120-V120, but only the V120-containing dimers are secreted. This selective retention of V0 homodimers indicates that the V region is required for formation and secretion of native FN dimers. In an analogous in vivo situation, we show that plasma FN also lacks V0-V0 dimers and consists of V0-V+ and V+-V+ combinations. Dissection of V region sequences by deletion mapping localizes the major site involved in DN dimer secretion to an 18-amino acid segment within V95. In addition, high levels of dimer secretion can be restored by insertion of V into a heterologous site 10 kD COOH terminal to its normal location. We discuss the potential role of intracellular protein-protein interactions in FN dimer formation.

Analysis of the internal nuclear matrix: Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.

Subunit structure of cell surface proteins: disulfide bonding in antigen receptors, Ly-2/3 antigens, and transferrin receptors of murine T and B lymphocytes.

The surface proteins of lymphocytes from spleen and thymus and several cultured lymphoid tumor lines were radioiodinated in situ, solubilized with Triton X-100, and examined for the presence of disulfide-bonded subunits by two-dimensional (intact, reduced) NaDodSO4/polyacrylamide gel electrophoresis. [Hynes, R. O. & Destree, A. (1977) Proc. Natl. Acad. Sci. USA 74, 2855-2859]. Few lymphocyte surface proteins were found to consist of disulfide-bonded subunits, and the most prominent of these could be identified. In normal B lymphocytes and B-lymphoma cells, IgD or IgM (or both) were the major disulfide-bonded proteins, and these were easily detectable, even without immunoprecipitation. In contrast, analysis of thymocytes and T-lymphoma cells did not reveal any protein resembling immunoglobulin in its chain structure. The major labeled thymocyte membrane protein consisting of disulfide-bonded subunits was identified as the Ly-2/3 antigen. It appeared to contain disulfide-bonded homodimers of Mr 35,000 (alpha 2) noncovalently associated with a second pair of homodimers of Mr 30,000 (beta 2). Peptide mapping showed these polypeptides to be homologous. A third disulfide-bonded homodimer, which was heterogeneous in apparent Mr, appeared to be part of the Ly-2/3 complex. All cultured T- and B-lymphoma lines examined were found to possess a major surface protein that appeared to be a disulfide-bonded homodimer of a polypeptide of Mr 95,000. This protein was identified as the receptor for transferrin. It is suggested that the presence of two or more subunits in cell surface receptors renders their ligand functionally bivalent, making ligand-induced receptor aggregation possible.