Regulation of BCR-dependent germinal center B-cell formation by HGAL and insight into its emerging myeloid ortholog, C1ORF150

Abstract

The specificity of cytokine and immunoreceptor signaling frequently depends upon receptor recruitment of select adaptor proteins and specifically engaged effectors. This review focuses on the orthologous adaptor proteins, HGAL and C1ORF150, and aims to provide insight into their respective modulation of lymphoid and myeloid cell signaling, formation, and function. HGAL acts predominantly within germinal center B cells as an important BCR signal transducer. Effects on BCR signalosome assembly involve HGAL's localization to the plasma membrane via its lipidation, initial interactions with SYK, the pY-phosphorylation of HGAL including its recruitment of GRB2, and HGAL engagement of PDZ-RhoGEF and RhoA signaling. At ligated BCRs, this includes HGAL(-GRB2) stimulation of SYK kinase, attenuation of calcium flux-dependent and NF-κB expression, promotion of cSMAC formation, and cytoskeletal remodeling associated with HGAL-attenuated cell migration. HGAL and partnered effectors also impact on DLBCL pathogenesis, and studies are summarized on HGAL's actions (using DLBCL and Burkitt lymphoma B cells) including cell migration effects, HGAL modulation of cytoskeletal components, and insightful HGAL transgenic mouse and xenograft models. For C1ORF150, its HGAL-homologous subdomains are considered, together with studies that demonstrate C1OR150's FcϵRI- and KIT-mediated expression and phosphorylation in primary human mast cells. Intriguingly, recent GWAS studies have identified a C1ORF150 in-frame splice variant that is strongly associated with urticaria. Candidate mechanisms via which the encoded "C1ORF150-Δexon2" isoform affects mast cell degranulation are considered, including FcϵR1 and/or KIT receptor connections, and candidate "myristoylation switch" mechanisms.

Keywords: B-cells; C1ORF150; HGAL; adaptor proteins; mast cells.

Figure 1

HGAL’s interactive domains and signaling partners within ligated BCRs, effects on cytoskeletal remodeling, and DLBCL and Burkitt lymphoma cell migration. (A) HGAL’s multi-fold interactive domains—upon BCR ligation within GC B cells, HGAL provides five sets of interactive motifs. These include N-terminal myristoylation and palmitoylation sites; four modulated central phospho-tyrosine (pY) motifs (with BCR-activated SYK as an indicated phosphorylating kinase); a validated DDpY107ENV binding site for GRB2 docking; a validated C-terminal PDZ-RhoGEF binding motif (PBM); and an H91 site demonstrated to bind FBXO10, (and associated Ub ligase). (B) HGAL, GRB2, and SYK interactions, and effects on BCR-upstream signaling and supramolecular activation complexes (pSMACs, cSMACs)—following BCR ligation, HGAL, as complexed with GRB2 and SYK has been demonstrated in *DLBCL and Burkitt lymphoma cell lines to activate SYK (left panel) while attenuating calcium flux and NF-kB activation and the actions of (p)BTK and (p)BLNK (center panel) (note, “*” designates DLBCL cell lines). These latter effects also depend upon HGAL’s pY107 GRB2 binding site. HGAL additionally supports the formation of peripheral and central pSMACs and cSMACs, with roles for HGAL-pY107 defined especially for the coalescing of BCR’s within cSMACs (right panel). (C) HGAL-mediated engagement of BCR-activated PDZ-RhoGEF pathways to DLBCL and Burkitt lymphoma cell cytoskeletal remodeling, including direct HGAL–cytoskeletal interactions. Right column: roles for HGAL’s C-terminal PDZ-RhoGEF binding domain are outlined in a propagating Rho/ROCK pathway to cytoskeletal remodeling. This includes the reinforcement of such signaling events due to increased levels of HGAL in DLBCL and Burkitt lymphoma cells (for details, see Section 5). Left column: HGAL additionally has been demonstrated to interact directly with select cytoskeletal components and to promote the formation of focal adhesions and stress fibers. (D) HGAL attenuation of DLBCL and Burkitt lymphoma cell migration (as mediated via a PDZ-RhoGEF, RhoA Rock pathway)—studies of DLBCL and Burkitt lymphoma cell line migration (in response to SDF1) are summarized. Variables are indexed as HGAL expression (+); HGAL knockdown (KD); IgM ligation of BCRs; and expressed HGAL constructs. Relative migration observed (migration chambers) is represented as +, ++, +++, ++++, +++++ (right column). (E) HGAL promotes DLBCL and Burkitt lymphoma cell line tumorigenesis—in vivo outcomes of xenograph studies (NOD/SCID mice) are summarized. DLBCL lines employed are *U2934 (HGAL negative, +/− HGAL ectopic expression) and Bjab (HGAL positive, +/− HGAL knockdown). Tumor mass and survival at d40 and d80 are summarized.

Figure 2

C1ORF150 and HGAL homology; prospective roles of C1ORF150 as an adaptor protein within FcϵR1, KIT, and EPOR/JAK2 complexes, and GWAS association of a C1ORF150 splice variant with urticaria. (A) hC1ORF150 and h-HGAL conserved motifs include C1ORF150’s N-terminal MG2NY predicted myristoylation site and four pY-modulated and similarly spaced sites. A shared consensus GRB2 binding site [GY89EN(I)] and tandem C-terminal pY sites in C1ORF150 share similar sequences (and relative positions) as HGAL’s pY128 and pY148 sites (as SYK docking sites in HGAL). Unlike HGAL, C1ORF150 also lacks any candidate site for palmitoylation or PDZ-RhoGEF binding. (B) Models outlining prospective roles for C1ORF150 within FcϵR1, KIT, and hEPOR/JAK2 membrane–proximal signaling complexes. Left panel: as a comparator, core components of the BCR are diagrammed, including the LYN, BCR alpha, beta chain, and SYK-mediated recruitment of HGAL, together with GRB2. Center panel: the mast cell FcϵR1 is diagrammed, together with KIT as a positive co-regulating factor for mast cell development, and activation. IgE/FcϵR1 induction of C1ORF150 transcript expression is also diagrammed. SYK and GRB2 as FcϵR1 signal transducers also are outlined. For KIT, SCF-induced phosphorylation of C1ORF150 at pY110 together with an induced de-phosphorylation at (p)Ser10 is also diagrammed. KIT-mediated pY modulation of RHEX is also depicted. Right panel: based on the EPO/EPOR-mediated pY phosphorylation of C1ORF150, its plasma membrane localization, and its HGAL-homologous motifs, an initial basic model is framed for C1ORF150’s coupling with EPOR/JAK2 signaling complexes. Type-1 JAK2-coupled receptors (lower box insert) among hematopoietic progenitor cells with elevated C1ORF150 expression also are indicated. (C) For the urticaria GWAS-associated C1ORF150 splice variant, rs56043070[A], clinical correlates in heterozygous and homozygous patients are summarized, and splice variant associated heightened activation and degranulation are diagrammed. (D) Isoforms of C1ORF150, and HGAL (translated from validated transcripts) including urticaria associated C1ORF150 “isoform B”. Upper panel: for C1ORF150, its urticaria-associated transcript-encoded protein is diagrammed including its predicted 20-residue deletion (encoded by exon-2). For this variant transcript, C1ORF150’s predicted myristoylation site is retained but with the conversion of an adjacent (p)Ser10 residue to Arg10. Lower panel: for HGAL, in isoform B, Arg10 interestingly becomes converted to Ser10. In Isoform C, HGAL’s palmitoylation site is deleted, with Arg 10 represented (rather than S10).