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Identity of the elusive IgM Fc receptor (FcmuR) in humans.

Kubagawa H, Oka S, Kubagawa Y, Torii I, Takayama E, Kang DW, Gartland GL, Bertoli LF, Mori H, Takatsu H, Kitamura T, Ohno H, Wang JY - J. Exp. Med. (2009)

Bottom Line: Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes.After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation.Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

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Tyrosine and serine phosphorylation of FcμR upon stimulation. (A and B) BW5147 T cells stably expressing human FcμR were incubated in the presence (+) or absence (−) of 100 µM pervanadate for 15 min (A) or with the preformed IgM immune complexes for the indicated time periods (min) at 37°C (B) before cell lysis. FcμR was immunoprecipitated from cleared lysates with anti-FcμR (HM14) or control (Cont.) mAb–coupled beads, resolved on SDS–10% PAGE under reducing conditions, transferred onto membranes, and immunoblotted with rabbit antibody specific for phosphoserine of PKC substrates along with HRP-labeled goat anti–rabbit Ig antibody (anti-PKC P-Ser) or with HRP-labeled antiphosphotyrosine mAb (anti–P-Tyr) before visualization by ECL. After dissociating blotted antibodies, membranes were reprobed with biotin-labeled anti-FcμR mAbs along with HRP-labeled SA (anti-FcμR). These experiments were performed at least three times. Mr is shown in kilodaltons.
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fig5: Tyrosine and serine phosphorylation of FcμR upon stimulation. (A and B) BW5147 T cells stably expressing human FcμR were incubated in the presence (+) or absence (−) of 100 µM pervanadate for 15 min (A) or with the preformed IgM immune complexes for the indicated time periods (min) at 37°C (B) before cell lysis. FcμR was immunoprecipitated from cleared lysates with anti-FcμR (HM14) or control (Cont.) mAb–coupled beads, resolved on SDS–10% PAGE under reducing conditions, transferred onto membranes, and immunoblotted with rabbit antibody specific for phosphoserine of PKC substrates along with HRP-labeled goat anti–rabbit Ig antibody (anti-PKC P-Ser) or with HRP-labeled antiphosphotyrosine mAb (anti–P-Tyr) before visualization by ECL. After dissociating blotted antibodies, membranes were reprobed with biotin-labeled anti-FcμR mAbs along with HRP-labeled SA (anti-FcμR). These experiments were performed at least three times. Mr is shown in kilodaltons.

Mentions: To determine whether these conserved Ser and Tyr residues are phosphorylated upon stimulation, FcμR+ BW5147 T cells were treated with a tyrosine phosphatase inhibitor, pervanadate, or with preformed IgM immune complexes to cross-link FcμR. The FcμR was immunoprecipitated from the lysates of resting or activated cells and analyzed by immunoblotting with antibodies specific for phosphotyrosine or the phosphoserine of PKC substrates. Phosphorylation of both serine and tyrosine residues was clearly demonstrated in pervanadate-treated cells but not in untreated cells (Fig. 5 A). Interestingly, the serine-phosphorylated FcμR migrated at ∼52 kD, whereas most of the tyrosine-phosphorylated FcμRs migrated at ∼60 kD and, to a lesser extent, at ∼52 kD. When these membranes were reprobed with anti-FcμR mAb specific for its extracellular epitope, we found that in resting cells FcμR was present as a major band of ∼60 kD, which is consistent with the Mr of the cell-surface FcμR (see Fig. 7), along with a minor band of ∼45 kD, but in pervanadate-treated cells the FcμR was resolved as a major band of ∼52 kD together with multiple minor species of various sizes. When FcμR was cross-linked with preformed immune complexes consisting of IgM and F(ab′)2 fragments of anti-μ mAb, phosphorylation of both serine and tyrosine residues of the ∼52 kD FcμR was also demonstrated as early as 3 min after ligation. The serine phosphorylation became more prominent at 30 min after ligation, whereas tyrosine phosphorylation was diminished by that time point (Fig. 5 B). In contrast to the effects seen with pervanadate treatment, tyrosine-phosphorylated ∼60-kD FcμR was not observed in the lysates of receptor-ligated cells. Reprobing of these membranes with anti-FcμR mAb revealed the presence of both ∼60- and ∼52-kD FcμR proteins as well as minor, but discrete, bands of ∼45 and ∼63 kD. Collectively, these findings suggest that the conserved serine and tyrosine residues seen in the cytoplasmic tail of FcμR are indeed potentially phosphorylated upon receptor ligation and that the phosphorylated FcμR protein migrates differently on SDS-PAGE compared with its unphosphorylated form.


Identity of the elusive IgM Fc receptor (FcmuR) in humans.

Kubagawa H, Oka S, Kubagawa Y, Torii I, Takayama E, Kang DW, Gartland GL, Bertoli LF, Mori H, Takatsu H, Kitamura T, Ohno H, Wang JY - J. Exp. Med. (2009)

Tyrosine and serine phosphorylation of FcμR upon stimulation. (A and B) BW5147 T cells stably expressing human FcμR were incubated in the presence (+) or absence (−) of 100 µM pervanadate for 15 min (A) or with the preformed IgM immune complexes for the indicated time periods (min) at 37°C (B) before cell lysis. FcμR was immunoprecipitated from cleared lysates with anti-FcμR (HM14) or control (Cont.) mAb–coupled beads, resolved on SDS–10% PAGE under reducing conditions, transferred onto membranes, and immunoblotted with rabbit antibody specific for phosphoserine of PKC substrates along with HRP-labeled goat anti–rabbit Ig antibody (anti-PKC P-Ser) or with HRP-labeled antiphosphotyrosine mAb (anti–P-Tyr) before visualization by ECL. After dissociating blotted antibodies, membranes were reprobed with biotin-labeled anti-FcμR mAbs along with HRP-labeled SA (anti-FcμR). These experiments were performed at least three times. Mr is shown in kilodaltons.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806608&req=5

fig5: Tyrosine and serine phosphorylation of FcμR upon stimulation. (A and B) BW5147 T cells stably expressing human FcμR were incubated in the presence (+) or absence (−) of 100 µM pervanadate for 15 min (A) or with the preformed IgM immune complexes for the indicated time periods (min) at 37°C (B) before cell lysis. FcμR was immunoprecipitated from cleared lysates with anti-FcμR (HM14) or control (Cont.) mAb–coupled beads, resolved on SDS–10% PAGE under reducing conditions, transferred onto membranes, and immunoblotted with rabbit antibody specific for phosphoserine of PKC substrates along with HRP-labeled goat anti–rabbit Ig antibody (anti-PKC P-Ser) or with HRP-labeled antiphosphotyrosine mAb (anti–P-Tyr) before visualization by ECL. After dissociating blotted antibodies, membranes were reprobed with biotin-labeled anti-FcμR mAbs along with HRP-labeled SA (anti-FcμR). These experiments were performed at least three times. Mr is shown in kilodaltons.
Mentions: To determine whether these conserved Ser and Tyr residues are phosphorylated upon stimulation, FcμR+ BW5147 T cells were treated with a tyrosine phosphatase inhibitor, pervanadate, or with preformed IgM immune complexes to cross-link FcμR. The FcμR was immunoprecipitated from the lysates of resting or activated cells and analyzed by immunoblotting with antibodies specific for phosphotyrosine or the phosphoserine of PKC substrates. Phosphorylation of both serine and tyrosine residues was clearly demonstrated in pervanadate-treated cells but not in untreated cells (Fig. 5 A). Interestingly, the serine-phosphorylated FcμR migrated at ∼52 kD, whereas most of the tyrosine-phosphorylated FcμRs migrated at ∼60 kD and, to a lesser extent, at ∼52 kD. When these membranes were reprobed with anti-FcμR mAb specific for its extracellular epitope, we found that in resting cells FcμR was present as a major band of ∼60 kD, which is consistent with the Mr of the cell-surface FcμR (see Fig. 7), along with a minor band of ∼45 kD, but in pervanadate-treated cells the FcμR was resolved as a major band of ∼52 kD together with multiple minor species of various sizes. When FcμR was cross-linked with preformed immune complexes consisting of IgM and F(ab′)2 fragments of anti-μ mAb, phosphorylation of both serine and tyrosine residues of the ∼52 kD FcμR was also demonstrated as early as 3 min after ligation. The serine phosphorylation became more prominent at 30 min after ligation, whereas tyrosine phosphorylation was diminished by that time point (Fig. 5 B). In contrast to the effects seen with pervanadate treatment, tyrosine-phosphorylated ∼60-kD FcμR was not observed in the lysates of receptor-ligated cells. Reprobing of these membranes with anti-FcμR mAb revealed the presence of both ∼60- and ∼52-kD FcμR proteins as well as minor, but discrete, bands of ∼45 and ∼63 kD. Collectively, these findings suggest that the conserved serine and tyrosine residues seen in the cytoplasmic tail of FcμR are indeed potentially phosphorylated upon receptor ligation and that the phosphorylated FcμR protein migrates differently on SDS-PAGE compared with its unphosphorylated form.

Bottom Line: Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes.After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation.Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

Show MeSH
Related in: MedlinePlus