This module will help you
Take-home message for this chapter: Binding of antigen to antigen-specific receptors on the outside of the plasma membrane changes gene expression (mRNA synthesis) in the nucleus. Concentrate on understanding the concepts without worrying about memorizing the names of every molecules (learn those in bold). Think about the challenge of sending different signals resulting in different responses through the same set of signal transducing molecules - for example, sometimes antigen activates lymphocytes, sometimes it kills them! Immunologists dream of being able to control this signaling in an antigen-specific manner: turning off autoimmune or rejection responses without interfering with normal adaptive immunity, or changing non-protective responses into protective ones.
Ligand-Receptor
Binding Alters Cell Function
Antigen Receptor Structure and Signaling
Other Receptor Pathways
Ligand-Receptor Binding Alters Cell Function
Signal transduction is the process of converting a signal from one form to another. Receptor is a general term for a molecule that receives signals from its ligand, the molecule it binds. Antigen is the ligand for BCR, peptide + MHC are the ligands for TCR. Membrane receptors all have extracellular domains that specifically bind ligands, transmembrane domains that span the plasma membrane, and cytoplasmic domains that participate in signal transduction. Molecules like TCR and BCR, which have very short cytoplasmic domains, are often associated with other signal transduction molecules like CD3 and IgaIgb. Although we are looking at signal transducers associated with the immune response, similar molecules function in other metabolic signaling.
The initial signal from antigen-binding to BCR and TCR results in a change in receptor clustering in the membrane. Multivalent antigens which cross-link BCR send strong signals to B cells, sometimes even activating them without T cell help to divide and synthesize IgM. Although B cells do not need antigen presentation to be activated, binding of BCR to an array of identical epitopes bound to antibody on Fc receptors (FcR) on macrophages and neutrophils efficiently stimulate B cell activation. Since each TCR binds only one antigen epitope, cross-linking probably involves multiple TCR binding multiple copies of a peptide on multiple MHC molecules on the APC or target cell.
The original clustering signal is transduced into chemical signals in the cytoplasm by the activation of receptor-associated protein kinases. Protein kinases of the immune system are enzymes which add phosphate groups to the serine, threonine, or tyrosine residues other proteins, often enzymes. Phosphorylation can activate some enzymes and inactivate others. Another result of phosphorylation is the creation of a binding site. Phosphorylated membrane proteins bind other proteins that are normally free in the cytoplasm. Binding increases the concentration of the cytoplasmic proteins and their ability to be phosphorylated.
Other enzymes called phosphatases remove the phosphate groups and reverse the signaling. In general, every signal is rapidly reversed in the absence of ligand. The cell is rapidly activated in the presence of antigen and just as rapidly returned to an inactive state when the antigen is removed.
Many of the signaling molecules involved in immune activation were discovered for their role in cancer. For example, a chicken tumor-causing virus called Rous sarcoma virus uses a gene called v-src (pronounced "vee-sark") to induce cell division in the host cells, making it easier for the virus to be copied. Since the presence of the gene causes cancer, it is called an oncogene. Oncogenes are often abnormally functioning normal genes involved in cell growth.
An important signaling molecule which binds phosphorylated membrane molecules is phospholipase C (PLC). PLC is an enzyme that cuts the membrane phospholipid phosphatidylinositol bisphosphate (PIP2 ) into two other signaling molecules, inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 diffuses away from the membrane and releases calcium from intracellular storage sites. Increased calcium levels activates the calcium-binding molecule calmodulin, which binds and activates other signaling molecules. DAG remains associated with the inner side of the plasma membrane and (with calcium) activates protein kinase C (PKC), which phosphorylates still other signaling molecules. This cascade of enzyme activations rapidly transmits the membrane binding signal through the cytoplasm.
The cytoplasmic signaling molecules can function with different receptors which send similar signals. For example, both antigen and growth factors signal lymphocytes to divide. In order for the signaling molecules to bind different receptors, adaptor proteins are needed. Adaptor proteins are not signaling molecules, but they connect the signaling enzymes with their substrates.
Small G proteins also link receptor tyrosine phosphorylation to cytoplasmic kinases. Small G proteins like Ras are inactive when they bind GDP and active when they bind GTP. Since small G proteins have phosphatase activity that converts GTP to GDP, they are normally inactive. Adaptor proteins bind small G proteins to the membrane receptors. Receptor-activated factors (GEFs) then displace GDP and allow GTP to bind and activate the small G protein. The small G protein then activates a cascade of protein kinases called MAP kinases, which phosphorylate transcription factors in the nucleus. Transcription factors bind the promoter region of the DNA preceding the coding region and promote RNA polymerase binding and synthesis on mRNA.
Both BCR and TCR are found on the plasma membrane with invariant signal transduction molecules: IgaIgb for BCR and CD3 for TCR. CD3 is composed of three dimers: gamma/epsilon (ge), delta/epsilon (de), and either two zetas (zz) or a zeta/eta (zh) heterodimer. [The g and d chains of CD3 are not the same molecules found in the gd TCR]. Gamma, delta, and epsilon chains have negatively charged transmembrane regions which form salt bridges with the positively charged transmembrane regions of TCR a and b chains. Most of the zeta chain molecule is cytoplasmic and transmembrane, with only a few amino acids exposed outside the plasma membrane.
The signal transduction molecules cluster in the plasma membrane with the antigen-specific receptors. If the signal transduction molecules cannot be made by the lymphocyte, no antigen receptors are expressed on the cell membrane. Both CD3 and IgaIgb have much longer cytoplasmic domains than BCR and TCR. They also have specific amino acid sequences, ITAMs (Immunoreceptor Tyrosine-based Activation Motifs), which become phosphorylated by receptor-associated tyrosine kinases.
When the antigen receptors cluster after binding antigen, src-family protein tyrosine kinases (PTKs) phosphorylate the ITAMS on the IgaIgb or the CD3. The activity of the src-family PTKs are regulated by two other protein kinases. Activation depends on CD45 (leukocyte common antigen) which is required for receptor-mediated activation of lymphocytes. Several specific src-family PTKs have been identified through diagnosis of people born with immune deficiencies resulting from defects in these enzymes.
Both T cells and B cells have co-receptors which enhance antigen signaling. The B cell co-receptors are CD19, CD21, and CD81 (TAPA-1). When antigen binds complement C3d, the antigen-complement complex binds the complement receptor CD21 (also called complement receptor CR2) as well as BCR. Cross-linking of BCR and CD21 induces phosphorylation of CD19 and increases sensitivity of B cells to antigen by 1,000-10,000 fold. Binding of CD4 or CD8 on the T cell to MHC on the APC increases cytoplasmic signaling in the T cell.
Once the ITAMS in the cytoplasmic domains of IgaIgb or CD3 have been phosphorylated, they can bind other PTKs (Syk in B cells or ZAP-70 in T cells). The cascade of cytoplasmic factors includes many of those we discussed above: PKC, IP3, DAG, calcium, small G proteins, MAP kinases, and transcription factors. Via this sequence of enzymatic events, new mRNA synthesis (note: Janeway incorrectly says gene synthesis in the heading 5-11) occurs as transcription factors bind the DNA. Several anti-rejection drugs, including cyclosporin A and FK506, block T cell activation by selectively interfering with the transcription regulator NFAT.
Peptides that activate T cells are called agonist peptides. However, some structurally related peptides (antagonist peptides) do not activate the T cells but prevent them from responding to agonist peptides. The clinical importance of antagonist peptides has been seen with HIV infection. HIV has a high mutation rate, which allows it to escape adaptive immunity. Mutant peptides have been shown to block lymphocyte activation by agonist peptides. Other peptides lead to only partial activation of the T cell and are called partial agonists. Partial agonists may signal the T cells to secrete cytokines but not to proliferate. This may happen when peptide binds TCR but co-receptor (CD4 or CD8) does not bind MHC.
Other receptors besides TCR and BCR have signal transduction ITAMs. The Fc receptor for IgG3 (FCg3R) on NK cells, macrophages, and neutrophils bind IgG3-coated antigen on pathogens or cell surfaces and signals the cells to kill in a process called antibody-dependent cell-mediated cytotoxicity (ADCC). Killer activatory receptors (KARs) on NK cells bind MHC and signal NK cells to kill virus-infected cells or tumor cells. The FcR for IgE (FceR1) on mast cells binds IgE that has not yet bound antigen. When antigen (allergens such as pollen) cross-links the IgE, the mast cells are signaled to release histamine and other molecules causing inflammation.
A family of inhibitory receptors on lymphocytes send "off" signals via Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs). Examples of these receptors are the FcgR IIB-1 and CD22 molecules on B cells, CTLA-4 on T cells, and killer inhibitory receptors (KIR) on NK cells. ITIMs bind phosphatases that dephosphorylate and thereby inactivate signaling molecules.
Other receptors are required for signals that influence leukocyte development and function. These include receptors for growth factors, cytokines, and death signals. Carefully regulated signaling results in homeostasis, the maintenance of leukocyte numbers within normal limits.
Lipopolysaccharide (LPS) and other microbial products can stimulate the release of the transcription factor NFkB in lymphocytes. They work by triggering a protease cascade that generates a ligand for the Toll-Like Receptor(TLR). Interleukin-1 (IL-1), a cytokine released by macrophages in response to microbial components, acts through the same or a very similar pathway; the cytoplasmic domains of IL-1R and Toll receptor are identical. Toll is interesting because it or very similar proteins have been shown to participate in defenses against infections in plants and Drosophila melanogaster as well as in mammals, suggesting it is one of the earliest parts of innate defense systems. NFkB is also required for HIV production in infected T cells.
Other cytokines signal by binding their specific receptors and triggering Janus kinases (JAKs) to phosphorylate and activate cytosolic STAT proteins (Signal Transducers and Activators of Transcription). STAT proteins then form dimers that migrate to the nucleus and initiate transcription.
The Fas receptor binds Fas ligand, becomes a trimer, and initiates signaling that results in activation of caspases and cleavage of DNA into 200 base pair fragments. (Binding of TNF to its receptor TNFR-1 can also induce caspase activation). This process occurs during apoptosis (programmed cell death) and is important for T cell mediated cytotoxicity and for homeostasis. Apoptosis can be detected using the TUNEL staining technique.
Expression of Fas during an immune response is believed to allow for regulation of immunity. Lymphocyte survival requires a balance between death-promoting (bax and bad) and death-inhibiting (bcl-2 and bcl-XL) genes. Signaling through the TCR and BCR is also crucial for maintaining homeostasis of T and B cells.
Practice Quiz
Pick the one BEST answer for each question by clicking on the letter of the correct choice.
1. An antigen binding signal at the membrane results in the mature B lymphocyte changing its
a. antigen-binding specificity.
b. color.
c. Ig V-D-J gene rearrangement.
d. gene expression.
e. signal transduction molecules.
2. Signal transduction is the process of converting
a. a B cell to a T cell.
b. a binding signal to a chemical signal.
c. a hapten to an antigen.
d. IgA to secretory IgA.
e. a kinase to a phosphatase.
3. A ligand is
a. a cytokine.
b. a molecule that specifically binds a receptor.
c. an antigen.
d. an enzyme.
e. all of the above are ligands.
4. A tyrosine kinase which is activated by antigen binding is found in the __________ of the BCr or TCR complex.
a. cytoplasmic domain
b. extracellular domain.
c. Ig superfamily domain.
d. transmembrane domain.
e. variable domain.
5. The ligand for TCR is
6. An oncogene is a gene that is associated with
a. apoptosis.
b. cancer.
c. ITIMs.
d. TCR and BCR signal transduction.
e. viruses.
7. Antigen binding to B cells is most effective at sending an activation signal to the B cell if it causes
a. antigen processing and presentation on Class II MHC.
b. BCR clustering.
c. BCR internalization.
d. inflammation.
e. opsonization.
8. An enzyme which puts a phosphate group on a protein molecule is called a
a. co-receptor.
b. ITAM.
c. kinase.
d. phosphatase.
e. receptor.
9. Gene expression does NOT necessarily involve
a. changes in a cell's activities (phenotype).
b. mRNA synthesis.
c. protein synthesis.
d. DNA synthesis.
e. transcription factors.
10. The signal transduction molecules associated with TCR are
11. The signal transduction molecules associated with BCR are
a. CD21 and CD81.
b. Iga and Igb
c. IgD and IgM.
d. ITAMs and ITIMs.
e. RAG-1 and RAG-2.
12. The second messenger IP3 increases the cytoplasmic concentration of
a. antigen.
b. calcium.
c. Class I MHC.
d. phosphate.
e. sodium.
13. DAG and IP3 are released from PIP2 by the action of
a. adaptor protein.
b. phospholipase C (PLC).
c. protein kinase C (PKC).
d. small G protein.
e. TdT.
14. Small G proteins (like Ras) convert GTP to GDP by their ___________ activity.
a. GEF.
b. kinase.
c. phosphatase.
d. polymerase.
e. protease.
15. Transcription factors
a. increase synthesis of mRNA.
b. increase synthesis of DNA.
c. inhibit synthesis of mRNA.
d. promote DNA phosphorylation.
e. synthesize mRNA.
16. An enzyme cascade is a
a. case where the enzyme catalyzes its own inactivation, like small G proteins.
b. pair of enzymatic reactions that have opposite effects, like kinases and phosphatases.
c. series of enzymatic reactions that result in cancer.
d. series of enzymatic reactions where the product of one reaction catalyzes the next reaction.
e. small waterfall.
17. Signal transduction complex associates with TCR in the membrane through
a. agonist peptides.
b. covalent bonds.
c. enzyme cascades.
d. reverse phosphorylation.
e. salt bridges.
18. If IgaIgb cannot be made, B cells
a. cannot express BCR.
b. cannot express Class II MHC.
c. express 1,000-fold less BCR than usual
d. synthesize CD3 and become T cells.
e. require 1,000-fold more antigen to be activated.
19. The immune system of a person who had a mutation in CD3 could NOT fight a viral hepatitis A infection by
a. blocking Hepatitis A virus from infecting liver cells with neutralizing IgG antibodies.
b. generating cytotoxic T cells to lyse infected liver cells
c. lysing virus-infected cells with NK cells.
d. phagocytosing complement-opsonized Hepatitis A virus.
e. Both 1 and 2 are correct.
20. Amino acid sequences in lymphocyte signal transduction complexes which are phosphorylated following antigen binding are called
21. An immune deficiency resulting from a defective PTK in the activation cascade in B cells would probably be characterized by
a. high numbers of circulating B cells.
b. high numbers of circulating lymphocytes.
c. high concentrations of plasma immunoglobulins.
d. low concentrations of plasma immunoglobulins.
e. low numbers of circulating T cells.
22. B cell co-receptor complex CD19, CD22, and CD81
a. allows B cells to be activated with 1,000-fold less complement-coated antigen.
b. allows B cells to be activated with 1,000-fold more complement-coated antigen.
c. decreases B cell expression of BCR.
d. increases B cell expression of BCR.
e. prevents B cell activation by self antigen.
23. The anti-rejection drugs cyclosporin A and FK506 block rejection of transplanted organs by interfering with
a. activation of a T cell transcription factor required for T cell activation.
b. antibody synthesis required for ADCC of transplanted cells.
c. CD3 expression.
d. MHC Class I expression.
e. processing of graft peptides and presentation on Class I MHC.
24. Antagonist peptides
a. fail to bind to T cells.
b. fully activate T cells.
c. interfere with T cell activation by agonist peptides.
d. partially activate T cells.
e. require partial agonist peptides to fully activate T cells.
25. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a process in which antibody-coated cells are killed by
a. the antibodies.
b. complement.
c. cytotoxic T cells.
d. cells with Fc receptors for IgG3.
e. cells with Fc receptors for IgE.
26. When IgE on mast cell FceR is cross-linked by, antigen, the mast cell responds by
a. apoptosis.
b. presenting the antigen to Th cells.
c. secreting IgE.
d. secreting histamine and other allergic mediators.
e. stimulating macrophage and neutrophil phagocytosis of the coated antigen.
27. Homeostasis is
29. Cells receive a death signal througha. macrophage activation by bacterial antigens.
b. programmed cell death.
c. the normal process of signal transduction.
d. the synthesis from all leukocytes from bone marrow stem cells.
e. the regulation of biological systems within normal limits.
a. bcl-2 receptor.
b. death receptor.
c. Fas.
d. Fas ligand.
e. STAT ligand.
30. The most important receptor through which lymphocytes receive life and death signals is
a. antigen receptor.
b. bcl-2 receptor.
c. Fas receptor.
d. FcR.
e. growth factor receptor.
Problem
What techniques could you use to determine whether small G proteins are required for signaling through the Fas Receptor?
