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B Baird

Publications and source records attributed to B Baird.

At least 37 records · Page 2Linked to original sources
PubMed1999

Membrane organization in immunoglobulin E receptor signaling.

The structure and dynamics of the plasma membrane are proposed to be critical for the initial steps of signal transduction by the high-affinity immunoglobulin E receptor. Recent experimental advances indicate that interactions between the high-affinity immunoglobulin E receptor and the tyrosine kinase Lyn with cholesterol- and sphingolipid-rich regions within the plasma membrane are important for receptor function. This accumulating evidence points to spatio-temporal control of immunoglobulin E receptor signaling by the organization of the plasma membrane; an attractive hypothesis is that ligand-dependent receptor aggregation causes the segregation of Lyn-containing ordered regions of the plasma membrane from disordered regions.

Animals↗
PubMed1999

Pharmacokinetics and safety of high-dose and extended-interval regimens of levofloxacin in human immunodeficiency virus-infected patients.

The pharmacokinetics of levofloxacin, administered in high doses and with extended dosing intervals, was studied in human immunodeficiency virus (HIV)-infected patients. Thirty patients received either 750 mg of the drug or a placebo once daily for 14 days, followed by 750 mg or 1,000 mg of the drug or a placebo three times weekly for an additional 14 days. Levofloxacin disposition was characterized by rapid oral absorption, with peak concentrations occurring approximately 1.5 h after dosing and elimination half-lives from 7.2 to 9.4 h. The overall incidence of any adverse effect was 70% (1,000 mg) to 95% (750 mg) for levofloxacin-treated patients and 71% for those taking the placebo. Levofloxacin pharmacokinetic parameters for HIV-infected patients were consistent with those observed in studies of healthy volunteers.

Administration, Oral↗
PubMed1998

Kinetics of multivalent antigen DNP-BSA binding to IgE-Fc epsilon RI in relationship to the stimulated tyrosine phosphorylation of Fc epsilon RI.

Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to Fc epsilon RI to stimulate cellular responses, although key features of the binding process are unknown. Fluorescence quenching can be used to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number of DNP groups per BSA is less than one. The binding data are well described by a transient hapten exposure model in which most of the DNP groups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measurement. Additional experiments indicate that, for suboptimal to optimal concentrations of DNP-BSA, most of the FITC fluorescence quenching on the cell surface is due to cross-linking events. With these concentrations at 15 degrees C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine phosphorylation of Fc epsilon RI. At 35 degrees C, the phosphorylation kinetics are biphasic during the time period in which cross-linking continues to increase. Our results establish a quantitative relationship between the time-course for cross-linking by multivalent Ag and Fc epsilon RI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.

Animals↗
PubMed1998

CXCR4 and CCR5 genetic polymorphisms in long-term nonprogressive human immunodeficiency virus infection: lack of association with mutations other than CCR5-Delta32.

Polymorphisms in the coding sequences of CCR5 and CXCR4 were studied in a group of human immunodeficiency virus (HIV)-infected long-term nonprogressors. Two different point mutations were found in the CXCR4 coding sequence. One of these CXCR4 mutations was silent, and each was unique to two nonprogressors. The well-described 32-bp deletion within the CCR5 coding sequence (CCR5-Delta32) was found in 4 of 13 nonprogressors, and 12 different point mutations were found scattered over the CCR5 coding sequence from 8 nonprogressors. Most of the mutations created either silent or conservative changes in the predicted amino acid sequence: only one of these mutations was found in more than a single nonprogressor. All nonsilent mutations were tested in an HIV envelope-dependent fusion assay, and all functioned comparably to wild-type controls. Polymorphisms in the CXCR4 and CCR5 coding sequences other than CCR5-Delta32 do not appear to play a dominant mechanistic role in nonprogression among HIV-infected individuals.

Female↗
PubMed1998

Stimulated release of fluorescently labeled IgE fragments that efficiently accumulate in secretory granules after endocytosis in RBL-2H3 mast cells.

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isothiocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but highly efficient accumulation of labeled IgE fragments in a pool of acidic peripheral vesicles that are visible by fluorescence microscopy after raising endosomal pH with ammonium chloride. Stimulation of cells containing these FITC-IgE fragments by aggregation of high affinity receptors for IgE (FcepsilonRI) or by Ca2+ ionophore and phorbol 12-myristate 13-acetate results in release of FITC fluorescence from the cells, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: it is prevented under conditions that prevent stimulated beta-hexosaminidase release, and these two processes exhibit the same antigen dose-dependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to FcepsilonRI at the cell surface causes internalization and delivery to the regulated secretory vesicles with a high efficiency similar to monomeric IgE-FcepsilonRI, but more rapidly. Binding of Cy3-modified IgE to FcepsilonRI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3-labeled vesicles can be observed by fluorescence microscopy without neutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with serotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-FcepsilonRI or addition of Ca2+ ionophore and phorbol 12-myristate 13-acetate, depletion of the Cy3 label from the intracellular vesicles is observed with confocal microscopy. These results provide strong evidence for the lysosomal nature of secretory granules in these cells. In addition, they provide the basis for a direct, real-time method for monitoring single cell degranulation.

Animals↗
PubMed1997

Evidence supporting a role for microfilaments in regulating the coupling between poorly dissociable IgE-Fc epsilonRI aggregates downstream signaling pathways.

Aggregation of Fc epsilonRI, the high-affinity receptor for IgE, on RBL-2H3 mast cells caused by reversible ligands such as multivalent antigen causes cellular responses that can be halted by subsequent addition of excess monovalent ligand. In contrast, Ca2+ and degranulation responses elicited by effectively irreversible streptavidin cross-linking of biotinylated IgE-Fc epsilonRI are not stopped by addition of excess biotin after stimulation is initiated. These results support previous conclusions based on studies with covalent oligomers of IgE that stable cross-links can continue to deliver stimulatory signals for extended periods of time. Dissociation measured in the presence of monovalent hapten reveals two populations of IgE-Fc epsilonRI cross-linked by multivalent antigen that differ in functional effectiveness. Aggregates with readily dissociable cross-links are normally responsible for triggering essentially all of the degranulation response, whereas aggregates with poorly dissociable cross-links apparently do not trigger this response. Treatment of RBL-2H3 cells with cytochalasin D, an inhibitor of actin polymerization, enhances downstream signaling and enables the less readily dissociable aggregates to stimulate Ca2+ and degranulation responses. Under these conditions, cytochalasin D does not affect hapten-mediated dissociation of multivalent antigen, nor does it prevent hapten from reversing tyrosine phosphorylation of Syk. Cytochalasin D alone causes tyrosine phosphorylation of a protein at approximately 75 kDa, and it reduces hapten-induced reversal of antigen-stimulated tyrosine phosphorylation of several other proteins. Taken together, these results indicate that stimulated actin polymerization normally regulates the coupling of aggregated Fc epsilonRI to downstream signaling pathways, and they provide an explanation for seeming discrepancies between responses to stable and reversible cross-links.

Actin Cytoskeleton↗
PubMed1997

Altered patterns of tyrosine phosphorylation and Syk activation for sterically restricted cyclic dimers of IgE-Fc epsilonRI.

Previous studies in our laboratory established that the symmetrical bivalent ligand, N,N'-bis-[[epsilon-(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L-cystin e ((DCT)2-cys), stably cross-links anti-2,4-dinitrophenyl-immunoglobulin E (IgE) bound to high affinity receptors Fc epsilonRI on the surface of RBL-2H3 cells, forming mostly cyclic dimers containing two IgE-Fc epsilonRI and two (DCT)2-cys (Posner et al. (1995) J. Immunol. 155, 3601-3609). These cyclic dimers do not trigger Ca2+ or degranulation responses under a variety of conditions. However, we find that the linearly cross-linked IgE-Fc epsilonRI formed at higher concentrations of (DCT)2-cys do trigger degranulation in the presence of cytochalasin D, an inhibitor of actin polymerization. We further investigated stimulation by (DCT)2-cys of the earliest known events in the functional response, i.e., tyrosine phosphorylation of the beta and gamma subunits of Fc epsilonRI. At the higher (DCT)2-cys concentrations corresponding to linear dimers and maximal degranulation, tyrosine phosphorylation of both beta and gamma are observed. At lower (DCT)2-cys concentrations where cross-linking is maximal and cyclic dimers are overwhelmingly dominant, only gamma tyrosine phosphorylation is observed. Cytochalasin D does not affect these phosphorylation patterns, but instead appears to enhance coupling to downstream signaling events. Phosphorylation of Syk occurs at the higher (DCT)2-cys concentrations in parallel with beta phosphorylation but does not occur in its absence at the lower (DCT)2-cys concentrations. These results suggest that cyclic dimers of IgE-Fc epsilonRI are sterically restricted such that they stimulate tyrosine phosphorylation of gamma but not beta, and this is not sufficient for Syk binding and/or activation.

Cell Line↗
PubMed1997

Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains.

The earliest known step in the activation of the high affinity IgE receptor, FcepsilonRI, is the tyrosine phosphorylation of its beta and gamma subunits by the Src family tyrosine kinase, Lyn. We report here that aggregation-dependent association of FcepsilonRI with specialized regions of the plasma membrane precedes its tyrosine phosphorylation and appears necessary for this event. Tyrosine phosphorylation of beta and gamma occurs in intact cells only for FcepsilonRI that associate with these detergent-resistant membrane domains, which are enriched in active Lyn. Furthermore, efficient in vitro tyrosine phosphorylation of FcepsilonRI subunits occurs only for those associated with isolated domains. This association and in vitro phosphorylation are highly sensitive to low concentrations of detergent, suggesting that lipid-mediated interactions with Lyn are important in FcepsilonRI activation. Participation of membrane domains accounts for previously unexplained aspects of FcepsilonRI-mediated signaling and may be relevant to signaling by other multichain immune receptors.

Cell Line↗
PubMed1996

Sustained T cell receptor-mediated Ca2+ responses rely on dynamic engagement of receptors.

We have investigated the functional advantage of surface-attached ligands for TCR-mediated cell activation with flow cytometric measurements of cytoplasmic Ca2+ changes in T cells after aggregation of TCR by soluble and bead-attached mAb. Conjugation of HPB-ALL human leukemia cells with cell-sized beads coated with anti-TCR mAb causes a stronger, more sustained Ca2+ response than that produced by the soluble form of the same mAb. Addition of a large excess of the soluble mAb subsequent to stimulation with the beads causes a marked reduction in the response of the bead-conjugated cells, but only limited disruption of the conjugates. Free (nonconjugated) cells, sampled simultaneously in this mixture, respond to the soluble mAb with a transient Ca2+ increase that declines with the same kinetics as the bead-conjugated cells after addition of the soluble mAb. Fab fragments of the anti-TCR mAb cause a similar reduction in the response of the bead-conjugated cells, and they do not stimulate free cells. Following the Fab-mediated decline in cytoplasmic Ca2+ of conjugated cells to near-baseline concentrations, the addition of a second, noncompetitive, anti-TCR mab causes a Ca2+ response that is substantially reduced in magnitude compared with that for the free cells. The results indicate that soluble and surface-attached ligands cause TCR-specific desensitization of the Ca2+ response. Surface-attached ligands are more effective than soluble ligands in sustaining signaling in T cells at least in part because they facilitate steady association and/or reassociation of TCR into the bound state in the surface contact area.

Aluminum Compounds↗
PubMed1996

The Fc segment of IgE influences the kinetics of dissociation of a symmetrical bivalent ligand from cyclic dimeric complexes.

As part of a systematic effort to determine the features of immunoglobulin E-receptor (IgE-Fc epsilon RI) aggregation that are critical for cellular activation, we used fluorescence to examine the dissociation of a soluble bivalent ligand, N, N'-bis[[epsilon-[(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L-cystine ((DCT)2-cys), from soluble bivalent IgE and its bivalent F(ab')2 and monovalent Fab' fragments. Cross-linking of Fab' fragments by (DCT)2-cys is limited to linear dimers, and we find that (DCT)2-cys dissociation from Fab' occurs with a single kinetic coefficient [(4.2 +/- 0.6) x 10-3 s-1] that corresponds to the lower of the two kinetic coefficients observed with the bivalent IgE [(4.7 +/- 0.7) x 10-2 s-1 and (4.4 +/- 0.3) x 10-3 s-1]. Similarly, the lower value is obtained for dissociation of (DCT)2-cys that is monovalently bound to IgE after incubation with a large excess of the ligand. (DCT)2-cys can bind to bivalent F(ab')2 fragments and form a variety of linear and cyclic aggregates, similarly to IgE, but, unlike IgE, we find that dissociation occurs with a single kinetic coefficient similar to that observed for Fab'. We find that IgE and its (Fab')2 fragments form highly stable cyclic dimer rings with two (DCT)2-cys. We demonstrate that the kinetic coefficients are independent of enhanced fluorescence quenching observed for bound sites in cyclic dimers. Together, the results show that the rate constant for breaking a linear cross-link formed by (DCT)2-cys is the same as that for dissociation of the monovalently bound (DCT)2-cys. Further, they show that opening of a bond in a dimer ring for the F(ab')2 fragment occurs with approximately the same dissociation rate constant as opening a bond in a linear cross-link. This rate constant is about three times smaller than that observed with IgE, suggesting that steric strain is caused by apposed Fc segments in cyclic IgE dimers. Such structural interference may affect the functional consequences of IgE-Fc epsilon RI aggregation on the cell surface.

Animals↗
PubMed1996

Constrained diffusion or immobile fraction on cell surfaces: a new interpretation.

Protein lateral mobility in cell membranes is generally measured using fluorescence photobleaching recovery (FPR). Since the development of this technique, the data have been interpreted by assuming free Brownian diffusion of cell surface receptors in two dimensions, an interpretation that requires that a subset of the diffusing species remains immobile. The origin of this so-called immobile fraction remains a mystery. In FPR, the motions of thousands of particles are inherently averaged, inevitably masking the details of individual motions. Recently, tracking of individual cell surface receptors has identified several distinct types of motion (Gross and Webb, 1988; Ghosh and Webb, 1988, 1990, 1994; Kusumi et al. 1993; Qian et al. 1991; Slattery, 1995), thereby calling into question the classical interpretation of FPR data as free Brownian motion of a limited mobile fraction. We have measured the motion of fluorescently labeled immunoglobulin E complexed to high affinity receptors (Fc epsilon RI) on rat basophilic leukemia cells using both single particle tracking and FPR. As in previous studies, our tracking results show that individual receptors may diffuse freely, or may exhibit restricted, time-dependent (anomalous) diffusion. Accordingly, we have analyzed FPR data by a new model to take this varied motion into account, and we show that the immobile fraction may be due to particles moving with the anomalous subdiffusion associated with restricted lateral mobility. Anomalous subdiffusion denotes random molecular motion in which the mean square displacements grow as a power law in time with a fractional positive exponent less than one. These findings call for a new model of cell membrane structure.

Animals↗
PubMed1996

Fc epsilon RI-mediated association of 6-micron beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling.

Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (Fc epsilon RI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6-micron beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses. The ligand-coated 6-micron beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand-coated Sepharose beads stimulate ample degranulation. Confocal fluorescence microscopy shows that the 6-micron beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that, beginning with the initial stages of bead engulfment, there is exclusion of many plasma membrane components from the 6-micron bead/cell interface, including p53/56lyn and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-Fc epsilon RI. The fluorescent lipid probe DiIC16 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DiIC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL-2H3 cells and the large Sepharose beads. Additional confocal microscopy analysis indicates that microfilaments are involved in the exclusion of plasma membrane components from the cell/bead interface. These results suggest that initiation of phagocytosis diverts normal signaling pathways in a cytoskeleton-driven membrane clearance process that alters the physiological response of the cells.

Animals↗
PubMed1996

Antigen-mediated IGE receptor aggregation and signaling: a window on cell surface structure and dynamics.

The high-affinity receptor for immunoglobulin E, Fc epsilon RI, serves as an archtype for multisubunit immunoreceptors that mediate cell activation in response to foreign antigens. Antigen-mediated aggregation of this receptor at the surface of mast cells and basophils initiates a biochemical cascade that uses nonreceptor tyrosine kinases as key participants in the earliest steps of this signal transduction process. Cross-linking of Fc epsilon RI with ligands of well-defined structure and valency has revealed detailed information about the fundamental requirements for functionally active receptor aggregates. Cross-linking-dependent changes in the interaction of these receptors with other cellular components have been characterized with biochemical and biophysical methods to develop a more complete view of signal initiation. Recent evidence suggests that this process involves the interaction of aggregated Fc epsilon RI with specialized plasma membrane domains that may localize important signaling molecules in the vicinity of aggregated receptors. Although these various studies were aimed toward understanding the operation of one cell surface receptor, they provide new insights into plasma membrane structure and dynamics that are generally relevant to the function of most nucleated mammalian cells.

Amino Acid Sequence↗
PubMed1995

Fluorescence resonance energy transfer reveals interleukin (IL)-1-dependent aggregation of IL-1 type I receptors that correlates with receptor activation.

Fluorescence resonance energy transfer (FRET) was used to investigate whether interleukin-1 (IL-1) causes the aggregation of IL-1 type I receptors (IL-1 RI) at the cell surface. For these experiments, a noncompetitive anti-IL1 RI monoclonal antibody, M5, was labeled separately with a donor probe, fluorescein isothiocyanate, or with an acceptor carbocyanine probe, Cy3. Donor-labeled M5 and acceptor-labeled M5 were simultaneously bound to transfected mouse IL-1 RI on either C-127 mouse mammary carcinoma cells or on Chinese hamster ovary (CHO)-K1 cells, and the ratio of acceptor emission at 590 nm to donor emission at 525 nm (excitation at 488 and 514 nm) was monitored with flow cytometry as an indicator of FRET. Addition of a saturating concentration of human IL-1 alpha at 22 degrees C causes a time-dependent increase in FRET for both cell lines that indicates IL-1-dependent self-association of IL-1 RI. Binding of the IL-1 receptor antagonist at 22 degrees C causes little or no FRET for both cell lines, indicating a correlation between receptor aggregation and the ability of the ligand to stimulate a functional response. When donor-labeled and acceptor-labeled Fab fragments of M5 are used to monitor FRET, IL-1 alpha causes efficient energy transfer in the CHO-K1 cells at 22 degrees C, but not at 4 degrees C. In contrast, IL-1 alpha causes much less FRET at 22 degrees C in C-127 cells when the M5 Fab fragments are used instead of the intact bivalent M5. In a striking parallel, IL-1 alpha-dependent activation of prostaglandin E2 production depends on the bivalent M5 antibody in the C-127 cells, but is independent of this monoclonal antibody in the CHO-K1 cells. These results provide a strong correlation between the ability of IL-1 to cause the aggregation of IL-1 RI and the stimulation of a functional response.

Animals↗
PubMed1995

Simultaneous cross-linking by two nontriggering bivalent ligands causes synergistic signaling of IgE Fc epsilon RI complexes.

We have used two bivalent ligands that bind IgE to study the relationship between the aggregation of receptors with high affinity for IgE (Fc epsilon RI) and the responses (receptor immobilization, Ca2+ influx, and degranulation) of rat basophilic leukemia (RBL-2H3) cells. One of these is a symmetric bivalent ligand, N,N'-bis[[epsilon-[(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L- cystine ((DCT)2-cys), which binds specifically to the combining sites of a mAb anti-DNP IgE and efficiently cross-links cell surface IgE, but does not trigger significant degranulation or increases in intracellular Ca2+. Several lines of evidence, including lateral mobility measurements, indicate that this ligand preferentially forms stable cyclic complexes containing two (DCT)2-cys and two IgE. The second ligand is a mAb anti-IgE, B1E3, which causes lateral mobility changes consistent with dimerized IgE-Fc epsilon RI and also does not trigger increases in intracellular Ca2+ or degranulation. The two ligands together trigger robust responses. In the presence of B1E3, (DCT)2-cys causes immobilization of IgE-Fc epsilon RI in a broad concentration range; in a more narrow concentration range, it is a potent stimulant of changes in both degranulation and Ca2+. We have compared the dose-response curves for cellular activation to simulated IgE aggregation curves, i.e., curves that predict the equilibrium IgE aggregate size distribution as a function of the (DCT)2-cys concentration. Our results indicate that maximal cellular activation occurs at a much higher (DCT)2-cys concentration than maximal IgE aggregation. When IgE aggregation is maximal, almost all aggregated IgE is in cyclic dimers. Thus, cyclic dimers appear to be functionally ineffective, even after they have been cross-linked by B1E3. Aggregated IgE-Fc epsilon RI that is effective in stimulating a cellular response may have particular structural or dynamic properties that allow critical interactions for initiating the signaling cascade.

Animals↗
PubMed1995

Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling.

Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we have isolated low-density, Triton X-100-insoluble membrane domains from RBL-2H3 mucosal mast cells that contain several markers common to caveolae, including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes a significant increase in the amount of p53/56lyn associated with these low-density membrane domains. Under our standard conditions for lysis, IgE-Fc epsilon RI fractionates with the majority of the solubilized proteins, whereas aggregated receptor complexes are found at a higher density in the gradient. Stimulated translocation of p53/56lyn is accompanied by increased tyrosine phosphorylation of several proteins in the low-density membrane domains as well as enhanced in vitro tyrosine kinase activity toward these proteins and an exogenous substrate. With a lower detergent-to-cell ratio during lysis, significant Fc epsilon RI remains associated with these membrane domains, consistent with the ability to coimmunoprecipitate tyrosine kinase activity with Fc epsilon RI under similar lysis conditions [Pribluda, V. S., Pribluda, C. & Metzger, H. (1994) Proc. Natl. Acad. Sci. USA 91, 11246-11250]. These results indicate that specialized membrane domains may be directly involved in the coupling of receptor aggregation to the activation of signaling events.

Animals↗
PubMed1995

Effects of subunit mutation on the rotational dynamics of Fc epsilon RI, the high affinity receptor for IgE, in transfected cells.

Erythrosin-labeled immunoglobulin E (IgE) and time-resolved phosphorescence anisotropy were used to monitor the rotational dynamics of transfected wild-type (alpha beta gamma 2) and four mutant Fc epsilon RI receptors in the monomeric and dimeric state on P815 cells. Erythrosin-IgE bound to Fc epsilon RI on cells transfected with either beta or gamma subunits with truncated COOH-terminal cytoplasmic segments exhibit faster rotational motion than when bound to Fc epsilon RI on cells transfected with wild-type subunits. Deletion of the NH2-terminal cytoplasmic segment of the beta subunit or the COOH-terminal cytoplasmic segment of the alpha subunit does not cause any significant change in the anisotropy decay. Dimers of IgE-receptor complexes formed with anti-IgE monoclonal antibody B1E3 exhibit substantially slower anisotropy decays for all the receptor constructs used, including a receptor construct that only contains the ectodomain of the alpha subunit anchored to Chinese hamster ovary (CHO) cell membranes through a lipid tail. This loss of rotational motion of dimeric IgE-Fc epsilon RI complexes may be due to nonspecific entanglement or to specific interactions involving IgE or the extracellular portion of alpha. The results suggest that the beta and gamma subunits of the tetrameric alpha beta gamma 2 receptor participate in interactions with other membrane components even in the absence of receptor aggregation. The loss of such interactions may be related to the functional impairments previously determined for these mutants.

Animals↗