B Cell Receptor-Associated Proteins

Background

About B Cell Receptor-Associated Proteins

B cell receptor-associated proteins are a group of molecules that are closely associated with the B cell receptor (BCR) complex. The BCR is a membrane-bound immunoglobulin (antibody) molecule expressed on the surface of B cells. It plays a crucial role in the recognition and binding of specific antigens, initiating the activation and immune response of B cells.

The BCR is composed of two main components: the membrane-bound immunoglobulin molecules and the associated signaling proteins. The signaling proteins that are associated with the BCR include Igα (CD79a) and Igβ (CD79b), which form a heterodimer known as the B cell antigen receptor complex (BCR complex). CD79a/CD79b heterodimeric unit contains three domains (extracellular, transmembrane, and intracellular) and is bound together with disulfide bonds formed between cysteine residues of its extracellular domains (Fig.1). These proteins are non-covalently associated with the immunoglobulin heavy and light chains of the BCR.

The BCR-associated proteins are critical for several key functions in B cell activation and immune responses. They serve as scaffolds for the recruitment and activation of signaling molecules, including kinases and adaptor proteins, which transmit the signals from the BCR to the nucleus. These signals regulate various cellular processes, such as B cell proliferation, differentiation, survival, and antibody production.

Notably, mutations or deficiencies in BCR-associated proteins, such as Igα and Igβ, can lead to impaired B cell function and immune deficiencies. For example, mutations in the Igα or Igβ genes can cause a rare primary immunodeficiency disorder called agammaglobulinemia, characterized by a lack of mature B cells and reduced antibody production.

Fig.1 Distinct function of CD79a/CD79b domains in B-cell receptor signaling. (Tkachenko A, et al., 2024)

Importance of BCR-Associated Proteins

BCR-associated proteins play a crucial role in B cell development, differentiation, and activation. Here are some key aspects highlighting their importance:

Key roles	Importance
B Cell Development	BCR-associated proteins, such as Ig chains (IgH and IgL), CD79a, and CD79b, are essential for the early stages of B cell development in the bone marrow. Ig chains are responsible for the assembly of the BCR and antigen recognition, while CD79a and CD79b form the BCR complex. Proper expression and function of these proteins are crucial for B cell maturation and selection processes, including V(D)J recombination, pre-BCR signaling, and positive/negative selection.
B Cell Activation and Signaling	BCR-associated proteins, including CD79a, CD79b, Lyn kinase, Syk kinase, and BLNK, play critical roles in BCR signaling and B cell activation. Upon antigen binding, BCR crosslinking leads to the phosphorylation of ITAMs within CD79a and CD79b by Lyn kinase. Phosphorylated ITAMs serve as docking sites for Syk kinase, which initiates downstream signaling cascades. BLNK acts as a scaffold protein, facilitating the assembly of signaling complexes and the recruitment of downstream signaling molecules. These signaling events trigger the activation of various pathways, such as the Ras-MAPK pathway, PI3K-Akt pathway, and NF-κB pathway, promoting B cell proliferation, survival, and differentiation.
Antibody Production	BCR-associated proteins are involved in the generation of antibody responses against specific antigens. BCR signaling, initiated by antigen recognition and BCR crosslinking, leads to B cell activation and subsequent differentiation into antibody-secreting plasma cells. Signaling pathways activated by BCR-associated proteins regulate the expression of genes involved in antibody class switching, affinity maturation, and the production of high-affinity antibodies.
Immune Responses and Defense	BCR-associated proteins are vital for mounting effective immune responses against pathogens. B cell activation and antibody production triggered by BCR signaling contribute to the clearance of pathogens, neutralization of toxins, and opsonization of microbes. BCR-associated proteins also participate in the formation of memory B cells, which provide long-term protection and rapid response upon re-exposure to antigens.
Autoimmunity and Tolerance	BCR-associated proteins, along with other regulatory molecules such as CD22 and CD5, help maintain self-tolerance and prevent autoimmune responses. CD22 acts as a negative regulator of BCR signaling, modulating the threshold for B cell activation and preventing excessive immune responses against self-antigens. CD5 influences BCR signaling and the balance between self-reactivity and responsiveness to foreign antigens, contributing to the regulation of autoimmunity.

In summary, BCR-associated proteins are indispensable for B cell development, differentiation, and activation. They enable B cells to recognize and respond to specific antigens, initiate signaling cascades, promote antibody production, and contribute to immune responses and defense mechanisms. Dysfunction or alterations in these proteins can lead to immunodeficiency, impaired immune responses, or dysregulated immune reactions, highlighting their vital role in maintaining a healthy immune system.

B Cell Receptor-Associated Proteins

Key Molecules and Functions Associated with BCR Activation

Key molecules	Functions
BANK1 (B-cell scaffold protein with ankyrin repeats 1)	Function: BANK1 acts as a scaffolding protein and is involved in signal transduction pathways. Role: BANK1 regulates B cell receptor signaling and influences B cell activation, differentiation, and antibody production.
BLK (B lymphocyte kinase)	Function: BLK is a non-receptor tyrosine kinase expressed in B cells. Role: BLK plays a role in B cell receptor signaling and contributes to B cell development, activation, and antibody production.
BLNK (B cell linker protein)	Function: BLNK acts as a scaffold protein, linking B cell receptor-associated signaling proteins. Role: BLNK is essential for BCR signaling, facilitating the recruitment and activation of downstream signaling molecules and regulating B cell activation and differentiation.
BTK (Bruton's tyrosine kinase)	Function: BTK is a cytoplasmic tyrosine kinase that is crucial for B cell receptor signaling. Role: BTK phosphorylates downstream signaling molecules, initiating intracellular signaling cascades and regulating B cell activation and antibody production.
CD19	Function: CD19 is a co-receptor protein expressed on B cells. Role: CD19 enhances B cell receptor signaling and amplifies B cell responses to antigen stimulation.
CD22	Function: CD22 is a transmembrane protein expressed on B cells. Role: CD22 functions as a negative regulator of B cell receptor signaling, contributing to the modulation of B cell activation and tolerance.
CD5	Function: CD5 is a cell surface glycoprotein expressed on B cells. Role: CD5 regulates B cell receptor signaling and influences B cell development, activation, and tolerance.
CD79A and CD79B	Function: CD79A and CD79B form a heterodimer known as the B cell antigen receptor complex (BCR complex). Role: CD79A and CD79B are essential for BCR signaling, transmitting signals from the BCR to intracellular signaling pathways and initiating B cell activation and antibody production.
CD81	Function: CD81 is a tetraspanin protein expressed on B cells. Role: CD81 is involved in B cell receptor signaling, B cell activation, and antibody production.
CR2 (Complement receptor 2)	Function: CR2 is a receptor for complement component C3d. Role: CR2 enhances B cell receptor signaling and facilitates the recognition of antigens opsonized with complement molecules.
FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, FCRL6 (Fc receptor-like proteins)	Function: FCRL proteins are a family of receptors with immunoregulatory functions. Role: FCRL proteins modulate B cell receptor signaling and influence B cell activation, differentiation, and antibody production.
FYN (Fyn tyrosine kinase)	Function: FYN is a Src family tyrosine kinase expressed in B cells. Role: FYN participates in B cell receptor signaling and contributes to B cell activation and antibody production.
LAX1 (Lymphocyte transmembrane adaptor 1)	Function: LAX1 is an adaptor protein expressed in B cells. Role: LAX1 is involved in B cell receptor signaling and influences B cell activation and differentiation.
Lyn (Lyn tyrosine kinase)	Function: Lyn is a Src family tyrosine kinase associated with the cytoplasmic tails of CD79A and CD79B. Role: Lyn initiates B cell receptor signaling by phosphorylating ITAMs within CD79A and CD79B, contributing to B cell activation and antibody production.
NFAM1 (Adaptor protein NFAT-activating molecule 1)	Function: NFAM1 is an adaptor protein expressed in B cells. Role: NFAM1 is involved in B cell receptor signaling and influences B cell activation, antibody production, and immune responses.
CD45	Function: CD45 is a transmembrane protein expressed on various immune cells, including B cells. Role: CD45 is a protein tyrosine phosphatase that regulates signaling events in B cell receptor signaling and influences B cell activation and differentiation.

These proteins, and others, collectively contribute to the complex signaling pathways and interactions involved in B cell receptor signaling, activation, and immune responses.

Key Signaling Pathways and Molecular Interactions Involved in BCR Activation

The activation of the B cell receptor (BCR) triggers a series of signaling pathways and molecular interactions that lead to B cell activation, proliferation, and antibody production. Here are some key signaling pathways and molecular interactions involved in BCR activation:

Key signaling pathways	Molecular interactions
BCR Crosslinking	When the BCR recognizes and binds to specific antigen molecules, it undergoes crosslinking, bringing multiple BCR complexes into close proximity.
Immunoreceptor Tyrosine-Based Activation Motifs (ITAMs)	The cytoplasmic tails of CD79a and CD79b, the signaling components of the BCR complex, contain ITAMs. ITAMs are phosphorylated by Lyn kinase upon BCR crosslinking.
Lyn Kinase Activation	Lyn kinase is associated with the cytoplasmic tails of CD79a and CD79b. Lyn kinase is activated by the phosphorylation of ITAMs.
Syk Kinase Recruitment and Activation	Phosphorylated ITAMs serve as docking sites for Syk kinase. Syk kinase is recruited to the phosphorylated ITAMs and becomes activated.
Phospholipase Cγ2 (PLCγ2) Activation	Activated Syk kinase phosphorylates and activates PLCγ2. Activated PLCγ2 generates secondary messengers, such as inositol trisphosphate (IP3) and diacylglycerol (DAG).
Calcium Signaling	IP3 binds to its receptor on the endoplasmic reticulum (ER), leading to the release of calcium ions (Ca2+) from the ER stores into the cytoplasm. Increased cytoplasmic calcium levels activate calcium-dependent signaling molecules, including protein kinases.
Protein Kinase Activation	Calcium-dependent protein kinases, such as protein kinase C (PKC), are activated by increased cytoplasmic calcium levels. PKC and other protein kinases phosphorylate downstream signaling molecules, amplifying the BCR signaling cascade.
BCR-Associated Scaffold Proteins	Scaffold proteins, such as B cell linker protein (BLNK), act as platforms for the assembly of signaling complexes. Phosphorylated ITAMs and activated kinases recruit scaffold proteins, facilitating the assembly of downstream signaling molecules.
Ras-MAPK Pathway	The activation of the BCR signaling cascade leads to the activation of Ras, a small GTPase. Ras activates the mitogen-activated protein kinase (MAPK) pathway, including extracellular signal-regulated kinases (ERKs). ERKs translocate to the nucleus and phosphorylate transcription factors, initiating gene expression involved in B cell activation and proliferation.
PI3K-Akt Pathway	Phosphoinositide 3-kinase (PI3K) is activated upon BCR signaling. Activated PI3K generates phosphoinositide signaling molecules, such as phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 recruits and activates the protein kinase Akt, which promotes cell survival, proliferation, and metabolic changes.
Nuclear Factor-κB (NF-κB) Pathway	BCR signaling activates the NF-κB pathway through multiple mechanisms. Activated NF-κB translocates to the nucleus and regulates the expression of genes involved in B cell activation, survival, and antibody production.
Transcriptional Regulation	BCR signaling regulates the expression of various transcription factors, such as NF-κB, AP-1, and others. These transcription factors coordinate the expression of genes involved in B cell activation, differentiation, and antibody production.

These signaling pathways and molecular interactions collectively contribute to BCR activation and subsequent B cell responses, including antibody production, class switching, affinity maturation, and memory B cell formation.

Fig.2 B-cell receptor signaling pathway. (Merolle MI, et al., 2018)

Diseases and Disorders Associated with Dysregulation of BCR-Associated Proteins

Dysregulation of BCR-associated proteins can contribute to various diseases and disorders. Here are some examples:

Immunodeficiencies

• Mutations in BCR-associated proteins, such as CD79a, CD79b, or Ig chains, can lead to primary immunodeficiencies.
• These immunodeficiencies result in impaired B cell development, reduced BCR signaling, and compromised antibody production, leading to increased susceptibility to infections.
• Examples include agammaglobulinemia, common variable immunodeficiency (CVID), and selective IgA deficiency.

Autoimmune Diseases

• Dysregulation of BCR-associated proteins can contribute to the development of autoimmune diseases, where the immune system mistakenly attacks self-tissues.
• Aberrant signaling through BCR-associated proteins can lead to the production of autoantibodies targeting self-antigens.
• Examples of autoimmune diseases associated with B cell dysregulation include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren's syndrome.

B Cell Malignancies

• Alterations in BCR-associated proteins can contribute to the development of B cell malignancies, including B cell lymphomas and leukemias.
• Mutations or chromosomal translocations involving BCR-associated proteins, such as IgH or CD79a/b, can result in constitutive BCR signaling and uncontrolled B cell proliferation.
• Examples include Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL).

Hyperactive B Cell Signaling

• Dysregulation of BCR-associated proteins can lead to hyperactive BCR signaling, resulting in excessive B cell activation and antibody production.
• This dysregulation can contribute to hypergammaglobulinemia, polyclonal B cell activation, and antibody-mediated disorders.
• Examples include hyper-IgM syndrome, certain forms of monoclonal gammopathy, and Waldenström macroglobulinemia.

Lymphoproliferative Disorders

• Dysregulated BCR signaling can contribute to the development of lymphoproliferative disorders characterized by abnormal B cell expansion.
• Aberrant BCR signaling can promote uncontrolled B cell proliferation and survival.
• Examples include lymphoproliferative disorders associated with chronic active Epstein-Barr virus (EBV) infection and hairy cell leukemia.

It's important to note that the dysregulation of BCR-associated proteins can occur through various mechanisms, including genetic mutations, chromosomal abnormalities, altered expression levels, and post-translational modifications. The resulting impact on B cell function and immune responses can vary depending on the specific protein involved and the nature of the dysregulation.

Case Study

Case 1: Jahn L, Hombrink P, Hassan C, et al. Therapeutic targeting of the BCR-associated protein CD79b in a TCR-based approach is hampered by aberrant expression of CD79b. Blood. 2015;125(6):949-958.

CD79b-specific T-cell clones K308 and S100 were assessed for their potency to recognize a variety of primary B-cell malignancies and malignant B-cell lines.

Clones K308 and S100 efficiently recognized HLA-A2–positive ALL cell lines (A). Clone S100 also recognized 4 HLA-A2–positive primary CLL samples (B). In addition, stimulation with primary HLA-A2–positive CLL and ALL samples resulted in strong IFN-γ production of clone K308 (C-D). No recognition of the HLA-A2–negative CLL sample ERV 8300 was observed (D). Coincubation of the ALL cell lines or primary HLA-A2–positive ALL and CLL samples with clone K308 resulted in efficient lysis of the malignant cells (E-F), indicating a cytotoxic effect of clone K308.

These results indicate efficient recognition of CD79b-expressing HLA-A2–positive B-cell malignancies by the CD79b-specific clones K308 and S100.

Fig.1 CD79b expression and shRNA knockdown confirm CD79b specificity of isolated T-cell clones.

Case 2: Huse K, Bai B, Hilden VI, et al. Mechanism of CD79A and CD79B support for IgM+ B cell fitness through B cell receptor surface expression. J Immunol. 2022;209(10):2042-2053.

To assess equivalent production of the rescue proteins, we linked CD79B to GFP using a 2A ribosome skipping peptide. Reintroduction of WT CD79B resulted in strong surface expression of CD79A, CD79B, and IgM, contrasting CD79B G137S, which led to low surface expression of the three BCR components (A). Importantly, the low surface expression levels observed with CD79B G137S were not due to lower retroviral transduction efficiency because both CD79B constructs showed similar GFP expression levels (A). In addition, CD79B mRNA expression levels were similar (B), whereas the total level of CD79B protein was higher for CD79B WT than CD79B G137S (C, D), suggesting that the mutant has reduced protein stability. However, the most striking difference was in protein maturation of CD79B (E).

These data demonstrate that CD79B protein governs the maturation and surface expression of both CD79A and IgM. The model that emerges from these results is that heterodimerization of CD79A and CD79B is required for proper protein maturation.

Fig.2 BCR surface expression and protein maturation in CD79B KO cells can be rescued by reintroducing CD79B.

References

• Merolle MI, Ahmed M, Nomie K, Wang ML. The B cell receptor signaling pathway in mantle cell lymphoma. Oncotarget. 2018;9(38):25332-25341.
• Tkachenko A, Kupcova K, Havranek O. B-cell receptor signaling and beyond: The Role of Igα (CD79a)/Igβ (CD79b) in normal and malignant B cells. International Journal of Molecular Sciences. 2024; 25(1):10.