Receptor Aggregation Research Articles

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp(63), Arg(116), and Lys(98) are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp(63) and Arg(116) form a salt bridge, whereas Lys(98) is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.

Divergent Intracellular Sorting of FcγRIIA and FcγRIIB2*

The human low affinity FcγRII family includes both the activating receptor FcγRIIA and the inhibitory receptor FcγRIIB2. These receptors have opposing signaling functions but are both capable of internalizing IgG-containing immune complexes through clathrin-mediated endocytosis. We demonstrate that upon engagement by multivalent aggregated human IgG, FcγRIIA expressed in ts20 Chinese hamster fibroblasts is delivered along with its ligand to lysosomal compartments for degradation, while FcγRIIB2 dissociates from the ligand and is routed separately into the recycling pathway. FcγRIIA sorting to lysosomes requires receptor multimerization, but does not require either Src family kinase activity or ubiquitylation of receptor lysine residues. The sorting of FcγRIIB2 away from a degradative fate is not due to its lower affinity for IgG and occurs even upon persistent receptor aggregation. Upon co-engagement of FcγRIIA and FcγRIIB2, the receptors are sorted independently to distinct final fates after dissociation of co-clustering ligand. These results reveal fundamental differences in the trafficking behavior of different Fcγ receptors.

Soluble HIV Tat Protein Removes the IL-7 Receptor α-Chain from the Surface of Resting CD8 T Cells and Targets It for Degradation

IL-7 signaling is essential to CD8 T cell development, activation, and homeostasis. We have previously shown decreased expression of the IL-7R alpha-chain (CD127) on CD8 T cells in HIV(+) patients and that this downregulation is mediated at least in part by the HIV Tat protein. We show in this study that CD127 has a prolonged t(1/2) in resting CD8 T cells and continuously recycles on and off the cell membrane. We also demonstrate soluble Tat protein significantly decreases the t(1/2) of CD127. Soluble Tat is taken up from the medium and accumulates in CD8 T cells with a peak of 6 h. Once inside the cell, Tat exits the endosomes during their normal acidification and enters the cytosol. Tat then translocates to the inner leaflet of the cell membrane, where it binds directly to the cytoplasmic tail of CD127, inducing receptor aggregation and internalization through a process dependent on microtubules. Tat appears to then target CD127 for degradation via the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is thus able to reduce IL-7 signaling and impair CD8 T cell proliferation and function.

Ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity

The ability to exogenously present cell-surface receptors in high-affinity conformations in a synthetic system offers an opportunity to provide host cells with protection from pathogenic toxins. This strategy requires improvement of the synthetic receptor binding affinity against its native counterpart, particularly with polyvalent toxins where clustering of membrane receptors can hinder binding. Here we demonstrate that reconstituted lipoprotein, nanometer-sized discoidal lipid bilayers bounded by apolipoprotein and functionalized by incorporation of pathogen receptors, provides a means to enhance toxin-receptor binding through molecular-level control over the receptor microenvironment (specifically, its rigidity, composition, and heterogeneity). Using a Foerster Resonance Energy Transfer (FRET)-based assay, we found that reconstituted lipoprotein incorporating low concentrations of ganglioside monosialotetrahexosylganglioside (GM1) binds polymeric cholera toxin with significantly higher affinity than liposomes or supported lipid bilayers, most likely a result of the enhanced control over receptor clustering provided by the lipoprotein platform. Using wide-area epifluorescence, we found that this enhanced binding capacity can be effectively utilized to divert cholera toxin away from populations of healthy mammalian cells. In summary, we found that reconstitutions of high-density lipoprotein can be engineered to include specific pathogen receptors; that their pathogen binding affinity is altered, presumably due to attenuation of receptor aggregation; and that these assemblies are effective at protecting cells from biological toxins.

The Calcium-binding Protein S100B Down-regulates p53 and Apoptosis in Malignant Melanoma

The S100B-p53 protein complex was discovered in C8146A malignant melanoma, but the consequences of this interaction required further study. When S100B expression was inhibited in C8146As by siRNA (siRNA(S100B)), wt p53 mRNA levels were unchanged, but p53 protein, phosphorylated p53, and p53 gene products (i.e. p21 and PIDD) were increased. siRNA(S100B) transfections also restored p53-dependent apoptosis in C8146As as judged by poly(ADP-ribose) polymerase cleavage, DNA ladder formation, caspase 3 and 8 activation, and aggregation of the Fas death receptor (+UV); whereas, siRNA(S100B) had no effect in SK-MEL-28 cells containing elevated S100B and inactive p53 (p53R145L mutant). siRNA(S100B)-mediated apoptosis was independent of the mitochondria, because no changes were observed in mitochondrial membrane potential, cytochrome c release, caspase 9 activation, or ratios of pro- and anti-apoptotic proteins (BAX, Bcl-2, and Bcl-X(L)). As expected, cells lacking S100B (LOX-IM VI) were not affected by siRNA(S100B), and introduction of S100B reduced their UV-induced apoptosis activity by 7-fold, further demonstrating that S100B inhibits apoptosis activities in p53-containing cells. In other wild-type p53 cells (i.e. C8146A, UACC-2571, and UACC-62), S100B was found to contribute to cell survival after UV treatment, and for C8146As, the decrease in survival after siRNA(S100B) transfection (+UV) could be reversed by the p53 inhibitor, pifithrin-alpha. In summary, reducing S100B expression with siRNA was sufficient to activate p53, its transcriptional activation activities, and p53-dependent apoptosis pathway(s) in melanoma involving the Fas death receptor and perhaps PIDD. Thus, a well known marker for malignant melanoma, S100B, likely contributes to cancer progression by down-regulating the tumor suppressor protein, p53.

Luminescent Hybrids Combining a Metal Complex and a Crown Ether – Receptors for Peptidic Ammonium Phosphates

AbstractHybrid compounds combining a luminescent aza crown ether and 1,4,7,10‐tetraazacyclododecane (cyclene) or bis(pyridin‐2‐ylmethylamine) (bpa) metal complexes were prepared. These synthetic receptors signal the presence of peptidic ammonium phosphates in buffered aqueous solution by changes in their emission intensity. Reversible phosphate ion coordination to the metal complex binding sites decreases the emission intensity of the phthalic ester fluorophore. Structurally suitable ammonium phosphates, such as the C‐terminal domain of RNA polymerase II or the protein kinase G target sequence, bind to the synthetic receptors by an additional intramolecular interaction between the ammonium ion and the aza crown ether. The binding of the ammonium ion to the aza crown ether increases the emission intensity of the fluorophore. In aqueous solution, this weak interaction can only occur within aggregates of phosphates and metal complex receptors of matching geometry;therefore, it allows the distinction of structurally related phosphorylated peptides in buffered aqueous solution.

Identification of Erbin Interlinking MuSK and ErbB2 and Its Impact on Acetylcholine Receptor Aggregation at the Neuromuscular Junction

Erbin, a binding partner of ErbB2, was identified as the first member of the LAP family of proteins. Erbin was shown at postsynaptic membranes of the neuromuscular junction (NMJ) or in cultured C2C12 myotubes (1) to be concentrated, (2) to regulate the Ras-Raf-Mek pathway, and (3) to inhibit TGF-beta signaling. In the CNS, Erbin interacts with PSD-95. Furthermore, agrin-MuSK signaling initiates formation of AChR aggregates at the postsynaptic membrane. In search of proteins interacting with MuSK, we identified Erbin as a MuSK binding protein. We verified the interaction of MuSK with Erbin, or both concomitantly with ErbB2 by coimmunoprecipitation, and we mapped the interacting epitopes between Erbin and MuSK. We demonstrated elevated mRNA levels of Erbin at synaptic nuclei and colocalized Erbin and MuSK at postsynaptic membranes. We identified several Erbin isoforms at the NMJ, all of which contained the MuSK binding domain. By knocking down Erbin, we observed agrin-dependent AChR aggregates on murine primary skeletal myotubes and C2C12 cells, and in the absence of agrin, microclusters, both of significantly lower density. Complementary, AChR-epsilon-reporter expression was reduced in myotubes overexpressing Erbin. We show that myotubes also express other LAP protein family members, namely Scribble and Lano, and that both affect physical dimensions of agrin-dependent AChR aggregates and density of microclusters formed in the absence of agrin. Moreover, MuSK-Erbin-ErbB2 signaling influences TGF-beta signaling. Our data define the requirement of Erbin on the cross talk between agrin and neuregulin signaling pathways at the NMJ.

Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

Mutations in MUSK causing congenital myasthenic syndrome impair MuSK–Dok-7 interaction

We describe a severe congenital myasthenic syndrome (CMS) caused by two missense mutations in the gene encoding the muscle specific receptor tyrosine kinase (MUSK). The identified MUSK mutations M605I and A727V are both located in the kinase domain of MuSK. Intracellular microelectrode recordings and microscopy studies of the neuromuscular junction conducted in an anconeus muscle biopsy revealed decreased miniature endplate potential amplitudes, reduced endplate size and simplification of secondary synaptic folds, which were consistent with postsynaptic deficit. The study also showed a striking reduction of the endplate potential quantal content, consistent with additional presynaptic failure. Expression studies in MuSK deficient myotubes revealed that A727V, which is located within the catalytic loop of the enzyme, caused severe impairment of agrin-dependent MuSK phosphorylation, aggregation of acetylcholine receptors (AChRs) and interaction of MuSK with Dok-7, an essential intracellular binding protein of MuSK. In contrast, M605I, resulted in only moderate impairment of agrin-dependent MuSK phosphorylation, aggregation of AChRs and interaction of MuSK with Dok-7. There was no impairment of interaction of mutants with either the low-density lipoprotein receptor-related protein, Lrp4 (a co-receptor of agrin) or with the mammalian homolog of the Drosophila tumorous imaginal discs (Tid1). Our findings demonstrate that missense mutations in MUSK can result in a severe form of CMS and indicate that the inability of MuSK mutants to interact with Dok-7, but not with Lrp4 or Tid1, is a major determinant of the pathogenesis of the CMS caused by MUSK mutations.

Contrasting requirements for DAP12 in receptor-mediated mast cell activation (86.4)

Abstract By inducing FcϵRI aggregation, antigen/IgE promotes mast cell cytokine production; a response markedly enhanced by specific TLR agonists. Critical to the ability of FcϵRI to activate mast cells is the recruitment of the tyrosine kinase Syk via the FcϵRIγ chains. Recent reports in other cell types have indicated that TLR’s may alternatively signal via the adaptor molecule DAP12 which also has the ability to bind Syk following tyrosine phosphorylation. We therefore examined whether DAP12 similarly regulates TLR-mediated responses in mast cells. DAP12 was confirmed to be expressed in both human and mouse mast cells (BMMCs) and, following its phosphorylation by pervanadate treatment, DAP12 recruited Syk. Although the TLR agonists LPS, Pam3Cys, and MALP2 induced cytokine production and synergistically enhanced this response mediated by FcϵRI, they failed to increase basal DAP12 phosphorylation. Furthermore, no up-regulation or down-regulation of TLR-mediated responses, nor FcϵRI-mediated responses, was observed in DAP12-/- BMMCs. In contrast, DAP12 phosphorylation and Syk recruitment were observed in mast cells following concanavalin A (Con A)-induced aggregation of mannose-glycosylated receptors, and these responses, together with ConA-induced degranulation, were substantially reduced in the DAP12-/- BMMCs. These data show that in mast cells DAP12 does not regulate TLR- or FcϵRI-mediated responses, but is required for responses elicited by mannose-glycosylated receptors.

Keeping Signaling in Check

Cytokine signaling is triggered by a hormone-induced receptor aggregation process. IL-13 employs an unconventional sequence of steps for assembling its signaling complex to trigger activation and for turning it off. Both these processes involve some unusual molecular recognition features, as discussed by Lupardus et al. (2010).

F-Actin Binding Regions on the Androgen Receptor and Huntingtin Increase Aggregation and Alter Aggregate Characteristics

Protein aggregation is associated with neurodegeneration. Polyglutamine expansion diseases such as spinobulbar muscular atrophy and Huntington disease feature proteins that are destabilized by an expanded polyglutamine tract in their N-termini. It has previously been reported that intracellular aggregation of these target proteins, the androgen receptor (AR) and huntingtin (Htt), is modulated by actin-regulatory pathways. Sequences that flank the polyglutamine tract of AR and Htt might influence protein aggregation and toxicity through protein-protein interactions, but this has not been studied in detail. Here we have evaluated an N-terminal 127 amino acid fragment of AR and Htt exon 1. The first 50 amino acids of ARN127 and the first 14 amino acids of Htt exon 1 mediate binding to filamentous actin in vitro. Deletion of these actin-binding regions renders the polyglutamine-expanded forms of ARN127 and Htt exon 1 less aggregation-prone, and increases the SDS-solubility of aggregates that do form. These regions thus appear to alter the aggregation frequency and type of polyglutamine-induced aggregation. These findings highlight the importance of flanking sequences in determining the propensity of unstable proteins to misfold.

K-Space Image Correlation Spectroscopy of Quantum dot Labeled T Cell Receptors Characterizes their Nanoscale Clustering in Living Cells

Changes in distribution of membrane receptor organization are used by cells to modulate the dynamic range of their responses to environmental cues. Consequently, it is important to develop experimental methods that can accurately measure receptor transport and aggregation. We used quantum dot (QD) labeling of T cell receptors (TCR) and a recently developed technique, k-space image correlation spectroscopy (kICS) to characterize TCR state as a function of cell differentiation. We developed kICS to measure transport coefficients of fluorescently labeled membrane proteins while taking into account nanoparticle emission blinking. We use kICS to measure T cell receptor (TCR) aggregation in live cells by characterizing quantum dot (QD) blinking and distribution on the cell surface. 2C TCR transgenic cells in culture were observed from the naive state to 12 days after activation by antigen. Cells were labeled with biotin-MHC/peptide to bind the TCR with relatively high avidity, followed by streptavidin-quantum dots. They were then imaged on an emCCD-equipped microscope and kICS analysis was applied. Spatial intensity fluctuations in an image measured the clustering of receptors on the 100s of nm length scale. Changes in the intensity correlation function of the blinking QDs characterized clustering on the 10s of nm length scale. We also used kICS to measure changes in TCR diffusive transport. When T cells exhibited maximum activity 3-4 days after exposure to antigen, the degree of their TCR aggregation on both length scales was significantly higher than that of naive cells, while TCR diffusion was a minimum. This new technology has powerful applications as it can be applied to just a few cells and we will show that it is able to detect changes in receptor organization of cells in vivo.

New role for Agrin in T cells and its potential importance in immune system regulation

Agrin plays a crucial role in the maintenance of the neuromuscular junction. However, it is expressed in other tissues as well, including T lymphocytes, where cell activation induces its expression. Agrin from activated T cells has the capacity to induce aggregation of key receptors and to regulate signalling. Interestingly, T cells isolated from patients with systemic lupus erythematosus over-express Agrin and its co-stimulation with the T cell receptor enhances production of pathogenic cytokines. These early studies point to an important function for Agrin in T cell biology and make the case for a more thorough and systematic investigation into its role in the immune system.

Distribution and Dynamics of RBL IgE Receptors (FcɛRI) Quantitatively Observed on Planar Ligand-Presenting Surfaces

There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. When T cells, B cells, or mast cells bind to monovalent ligands on fluid supported lipid bilayers, receptor clustering, signaling, and receptor redistribution into immunological synapses follow. We have quantitatively studied the kinetics of redistribution of IgE receptors (FcɛRI) on RBL-2H3 mast cell surfaces. To separate the kinetics of receptor redistribution from cell spreading, the initial cell-substrate contact time was precisely defined (±50 ms) by micropipette cell manipulation. Using total internal reflection fluorescence, the distribution and dynamics of receptor clusters were imaged. We find strong quantitative evidence that initial receptor aggregation occurs at cellular protrusions, which are visible bright spots/regions on cells contacting ligand-free surfaces. The initial size of these regions is independent of the substrate and the presence or absence of ligand, and they were found to be randomly distributed over the interfacial contact area. Using a finite-element diffusion model, we found that the initial rate of accumulation of receptors at the protrusions is consistent with diffusion-limited trapping with D ∼10−1 μm2/s. At longer timescales, individual clusters on ligand-bearing membranes were observed to move with both a diffusive and a directed component of motion; clusters eventually coalesced near the center of the contact region (∼ 1 minute). The dynamics of the early cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-ACO4-94AL85000.

Distribution of pathogenic androgen receptor aggregations is influenced by heat shock factor-1 in model mouse of spinal and bulbar muscle atrophy (SBMA)

Distribution of pathogenic androgen receptor aggregations is influenced by heat shock factor-1 in model mouse of spinal and bulbar muscle atrophy (SBMA)

Modeling Multivalent Ligand-Receptor Interactions with Steric Constraints on Configurations of Cell-Surface Receptor Aggregates

We use flow cytometry to characterize equilibrium binding of a fluorophore-labeled trivalent model antigen to bivalent IgE-Fc εRI complexes on RBL cells. We find that flow cytometric measurements are consistent with an equilibrium model for ligand-receptor binding in which binding sites are assumed to be equivalent and ligand-induced receptor aggregates are assumed to be acyclic. However, this model predicts extensive receptor aggregation at antigen concentrations that yield strong cellular secretory responses, which is inconsistent with the expectation that large receptor aggregates should inhibit such responses. To investigate possible explanations for this discrepancy, we evaluate four rule-based models for interaction of a trivalent ligand with a bivalent cell-surface receptor that relax simplifying assumptions of the equilibrium model. These models are simulated using a rule-based kinetic Monte Carlo approach to investigate the kinetics of ligand-induced receptor aggregation and to study how the kinetics and equilibria of ligand-receptor interaction are affected by steric constraints on receptor aggregate configurations and by the formation of cyclic receptor aggregates. The results suggest that formation of linear chains of cyclic receptor dimers may be important for generating secretory signals. Steric effects that limit receptor aggregation and transient formation of small receptor aggregates may also be important.

Bortezomib Enhances the Efficacy of Fulvestrant by Promoting the Aggregation of the Estrogen Receptor in the Cytoplasm.

Abstract Background: Aromatase inhibitors (AIs) are standard treatment for Estrogen Receptor (ER)+ breast cancers in post-menopausal women where the main source of estradiol comes from adipose tissue when the aromatase enzyme converts androgens to estrogens. However, in premenopausal women, functioning ovaries flood the body with estrogens, and aromatase inhibitors used alone offer no therapeutic benefit. In addition to tamoxifen and aromatase inhibitors, estrogen receptor down-regulators, are a third type of anti-estrogen. The first of this class to be FDA approved is Fulvestrant, which acts by promoting the proteosomal degradation of the ER. Like tamoxifen, fulvestrant binds directly to the ER but while tamoxifen has both antagonist and agonist effects on the ER, fulvestrant is a pure antagonist. Other important advantages of fulvestrant over tamoxifen are that 1) fulvestrant prevents the ER from binding DNA, 2) fulvestrant is not linked to increased risk of endometrial cancer, 3) fulvestrant promotes permanent degradation of the ER.The molecular machinery leading to the degradation of the ER in the nucleus following fulvestrant treatment is well described and correlates with its ubiquitination in the nucleus. A less well-recognized mechanism, is fulvestrant’s ability to promote the aggregation of the newly synthesized ER in the cytoplasm. Understanding that protein aggregates are toxic when not eliminated by the proteasome, we took advantage of this effect of fulvestrant to ask whether combining fulvestrant with the proteasome inhibitor Bortezomib could enhance the efficacy of Fulvestrant.Results: We found that bortezomib enhances the aggregation of the ER in the cytoplasm following treatment with fulvestrant. Further, these aggregates were found to be insoluble and to activate the unfolded protein response (UPR), a stress response that leads to cell death. Further, bortezomib is able to prevent the activation of cytoprotective responses linked to the acquisition of fulvestrant resistance. Furthermore, in a breast cancer mouse model of tamoxifen resistance, the combination induced tumor regression.Conclusion: We conclude that adding bortezomib to fulvestrant enhances its efficacy by taking advantage of a previously poorly recognized mechanism–fulvestrant’s induction of ER aggregation in the cytoplasm. Further, our data suggest that this strategy will block the ability of cells to acquired resistance to fulvestrant. Our group has developed a clinical trial that will test this combination. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5142.

Extracellular Neurotransmitter Receptor Clustering: Think Outside the Box

Postsynaptic submembrane scaffolds cluster neurotransmitter receptors through intracellular protein-protein interactions. Growing evidence supports the view that extracellular factors can be almost as important to trigger synaptic receptor aggregation.

Immunoliposomes Incorporated with Humanized Monoclonal Antibody, Milatuzumab, Induce Cell Death in CLL by Retention of the CD74 Receptor On the Surface of B Cells.

Abstract 721Chronic lymphocytic leukemia (CLL) is a progressive disease for which most patients require treatment. Therapy with monoclonal antibodies (mAbs) such as rituximab (anti-CD20) and alemtuzumab (anti-CD52) have improved the outcomes of patients with CLL; however, patients display varied levels of response to these agents and many develop resistance. We describe here a humanized mAb, milatuzumab (Immunomedics, Inc.), against CD74. The CD74 antigen has several ligands, including macrophage inhibitory factor, MIF, whose binding promotes internalization and nuclear localization with concomitant activation of NF-κB. Milatuzumab is currently in clinical trials in multiple myeloma and CLL; however, the mechanism by which this antibody induces apoptosis is still unknown. We demonstrate surface expression of CD74 on CLL and circulating normal CD19+ B cells, but no expression on T cells. Milatuzumab-induced cytotoxicity occurs very rapidly, with 70% cell survival at 4 h and 45% at 24 h, which is superior to that observed with the CD20 antibody rituximab. No toxicity was detected in NK and T cells treated with milatuzumab. The mode of cell death induced by milatuzumab was found to be independent of caspase activation, with no detectable caspase-3 processing, PARP cleavage, or inhibition of death with the pan-caspase inhibitor, BocD-fmk. In vitro, the addition of a-Fc crosslinking antibody is necessary for milatuzumab-induced cell death. However, previous reports noted milatuzumab does not mediate antibody-dependent cellular cytotoxicity (ADCC) in lymphoma cell lines and this was verified in CLL using peripheral blood mononuclear cells, NK cells, and granulocytes as effector cells. Thus, milatuzumab-mediated death appears to be mediated directly through interaction with CD74 and effects on signal transduction. We further investigated the role of anti-Fc crosslinking antibody in mediating milatuzumab-induced cell death, and found that upon milatuzumab treatment in the presence of anti-Fc, CLL cells aggregated in culture. Furthermore, the mean fluorescent intensity (MFI) of CD74 on the cell surface increased, which did not occur following treatment with anti-Fc alone, milatuzumab alone, or anti-Fc along with other anti-B-cell mAbs such as rituximab. This suggests that milatuzumab, when crosslinked, promotes the maintenance of CD74 on the cell surface and thereby prevents receptor internalization and subsequent signaling. However, association with Fc receptors on other cells in the microenvironment may not be sufficient to mediate this effect, as indicated by the lack of ADCC. Furthermore, in vitro co-culture of NK cells in the presence of milatuzumab did not induce the same cellular aggregation and elevated CD74 surface expression as seen with the anti-Fc antibody. These results indicate that milatuzumab treatment in vivo may not be as efficient as suggested by the in vitro studies without a sufficient method to sequester CD74 on the cell surface. We tested this hypothesis by incorporating milatuzumab into liposomes (Mila-ILP). Our results show that this conjugate mediates the same receptor aggregation on the cell surface as milatuzumab combined with anti-Fc crosslinking antibody. This effect was not evident with incorporation of irrelevant immunoglobulin into liposomes (IgG-ILP). Furthermore, Mila-ILP induced the same degree of cell death as observed previously with in vitro antibody crosslinking. Therefore, the incorporation of milatuzumab into liposomes induces cell death independent of other cell types in the microenvironment. Together, these data describe a mechanism of milatuzumab-induced cytotoxicity in CLL, and encourage the clinical application of milatuzumab-immunoliposome-based therapy in CLL and other CD74-positive lymphoid malignancies. Disclosures:Goldenberg:Immunomedics: Employment, Equity Ownership.