6-1. Binding of antigen leads to clustering of antigen receptors on lymphocytes

All cell-surface receptors that have a signaling function are either transmembrane proteins themselves, or form parts of protein complexes that link the exterior and interior of the cell. Many receptors undergo a change in protein conformation on binding their ligand. In some types of receptor, this conformational change opens an ion channel into the cell and the resulting change in the concentration of important ions within the cell acts as the intracellular signal, which is then converted into an intracellular response. In other receptors, the conformational change affects the cytoplasmic portion of the receptor, enabling it to associate with and activate intracellular signaling proteins and enzymes.

The antigen receptors on lymphocytes transmit a signal when they bind a ligand that causes them to cluster together on the cell surface. The requirement for receptor clustering was first shown experimentally in somewhat artificial systems by using antibodies against the extracellular portion of the receptor to mimic antigen binding. Antibodies specific for the B-cell receptor or the T-cell receptor activate signaling by inducing clustering of the receptor complexes. This is a very convenient system for the analysis of early events after activation, as all the cells in a sample will be stimulated at the same time, making the course of the response easier to follow.

Antigen receptor clustering occurs when the receptors are cross-linked to each other. The importance of cross-linking was shown by comparing the response to stimulation with antibody F(ab′)₂ fragments, which have two binding sites, and with Fab fragments, which have only one (see Fig. 3.3). On lymphocytes treated with Fab fragments the antigen receptors do not cluster and the cells make no response, whereas on lymphocytes treated with F(ab′)₂ fragments the receptors become dimerized and the cells respond, although they may respond only weakly. The response is strongest when the F(ab′)₂ cross-linked dimers are further clustered using anti-immunoglobulin sera directed against the F(ab′)₂ fragments. The extensive cross-linking of the antigen receptors that then occurs delivers a very strong signal to the cell (Fig. 6.1).

Figure 6.1

Cross-linking of antigen receptors is the first step in lymphocyte activation. The requirement for receptor cross-linking is illustrated by the use of anti-immuno-globulin antibodies to activate the B-cell antigen receptor (BCR). As shown in the left (more...)

How antigen receptors are clustered in vivo when B and T cells encounter their specific antigens is not yet completely understood. T-cell receptors are presumed to undergo clustering in response to contact with another cell surface bearing multiple copies of the specific MHC:peptide complex they recognize. As we will see in Chapter 8, the T-cell receptors become involved in an organized cluster with other cell-surface signaling molecules, but the details of this clustering remain poorly understood. B-cell receptors can be cross-linked by pathogens such as intact bacteria and viruses that have repetitive epitopes on their surfaces. Complex molecules that contain regularly repeated identical epitopes will have the same effect. However, it is still uncertain how B-cell receptors can be clustered by soluble monomeric antigens, such as most of the experimental antigens that immunologists use to study immune responses. An inability, or limited ability, of soluble monomeric antigens to induce receptor clustering may explain why the activation of naive B cells in response to these antigens depends on receiving activating signals from antigen-specific T cells. As we will see in Chapter 9, the binding of soluble monomeric antigen by the B-cell receptor triggers receptor-mediated endocytosis, but is not sufficient by itself to stimulate cell division and differentiation. However, receptor-mediated uptake allows the antigen to be processed and displayed as peptide fragments bound to MHC class II molecules at the cell surface. The B cell can then be recognized by an antigen-specific CD4 effector T cell, which can deliver activating signals that drive clonal expansion and differentiation.

Understanding how the binding of antigen leads to receptor clustering and signaling in lymphocytes is complicated by the diversity of antigen receptors and their ligands. In addition, as we will see in Section 6-8, co-receptors for antigen-linked molecules may also cluster with the receptor and contribute to the initiation of intracellular signaling. How ligand binding leads to receptor clustering and generates a signal is more clearly understood for some other simpler receptors, as we will see in the next section.

6-2. Clustering of antigen receptors leads to activation of intracellular signal molecules

Most of the receptors discussed in this chapter initiate intracellular signaling by the activation of protein tyrosine kinases, enzymes that affect the activity of other proteins by adding a phosphate group to certain tyrosine residues. The receptors for some growth factors provide the simplest example of this type of receptor. They have cytoplasmic domains that contain an intrinsic tyrosine kinase activity. These enzyme domains are normally inactive, but when brought together by receptor clustering they are able to activate each other by transphosphorylation (Fig. 6.2). Once activated, these tyrosine kinases can phosphorylate and activate other cytoplasmic signaling molecules.

Figure 6.2

Ligand binding to the growth factor receptor Kit induces receptor dimerization and transphosphorylation of its cytoplasmic tyrosine kinase domains. Kit (CD117) is a transmembrane protein with an external ligand-binding domain specific for stem-cell factor (more...)

The situation in the antigen receptors is somewhat more complex. As we will see later, they do not themselves have intrinsic tyrosine kinase activity. Instead, the cytoplasmic portions of some of the receptor components bind to intracellular protein tyrosine kinases, which are therefore known as receptor-associated tyrosine kinases. When the receptors cluster, these enzymes are brought together and act on each other and on the receptor cytoplasmic tails to initiate the signaling process as in the example above.

In the case of the antigen receptors, the first tyrosine kinases associated with the receptor are members of the Src (pronounced ‘Sark’) family of tyrosine kinases. The Src-family kinases are common components of signaling pathways concerned with the control of cell division and differentiation in vertebrates and other animals. The prototypic family member Src was initially discovered as the oncogene v-src which is responsible for the ability of the Rous sarcoma virus to produce tumors in chickens. This viral gene was subsequently shown to be a modified form of a normal cellular gene called c-src that the virus had picked up from its host cell at some time in the past. Several other common components of signaling pathways that regulate cell growth and division were also first discovered through their oncogenic action when mutated or removed from their normal controls.

The receptor-associated Src-family kinases play a key role in transducing signals across the lymphocyte membrane; their activation informs the cell interior that the receptor has encountered its antigen. But this is just the first step in a multistep process. When a cell is signaled by the binding of ligand to a kinase-coupled receptor, kinase activation initiates a cascade of intracellular signaling that transfers the signal to other molecules and eventually carries it to the nucleus.

6-3. Phosphorylation of receptor cytoplasmic tails by tyrosine kinases concentrates intracellular signaling molecules around the receptors

Phosphorylation of enzymes and other proteins by protein kinases is a common general mechanism by which cells regulate their biochemical activity, and has many advantages as a control mechanism. It is rapid, not requiring new protein synthesis or protein degradation to change the biochemical activity of a cell. It can also be easily reversed by the action of protein phosphatases, which remove the phosphate group. Many enzymes become active when phosphorylated and inactive when dephosphorylated, or vice versa; the activity of many of the protein kinases involved in signaling is regulated in this way.

Another and equally important outcome of protein phosphorylation is the creation of a binding site for other proteins. This does not alter the intrinsic activity of the phosphorylated molecule. In this case, phosphorylation is used as a tag, allowing the recruitment of other proteins that bind to the phosphorylated site. For example, many kinases involved in signaling are associated with the inner surface of the cell membrane and can act only in-efficiently upon their target proteins when these are free in the cytosol. Receptor activation and the phosphorylation of membrane-associated proteins can, however, create binding sites for these target proteins. Cytosolic proteins that bind to phosphorylated sites at the membrane are thus concentrated near to the kinase and can in their turn be phosphorylated and activated (Fig. 6.3). They can also, in some cases, be activated simply by binding to phosphotyrosine. This is an example of allosteric activation, as binding the phosphotyrosine leads to an alteration in their molecular conformation.

Figure 6.3

Receptor activation recruits cytosolic proteins to the signaling pathway. Receptor-associated protein kinases are localized at the inner surface of the cell membrane and cannot activate their cytosolic targets efficiently unless these are brought to the (more...)

Proteins can be phosphorylated on three classes of amino acid—on tyrosine, on serine or threonine, or on histidine. Each of these requires a separate class of kinases to add phosphate groups; only the first two are currently known to be relevant to signaling within the immune system. As we have seen, the early events of signaling, associated with the clustering of the antigen receptors, predominantly involve protein tyrosine kinases; the later events also involve protein serine/threonine kinases.

In antigen receptor signaling, the phosphotyrosines generated by tyrosine kinase action form binding sites for a protein domain known as an SH2 domain (Src homology 2 domain). This is found in many intracellular signaling proteins including the Src-family kinases, in which SH2 domains were first discovered. Binding of SH2 domains to phosphotyrosines is a crucial mechanism for recruiting intracellular signaling molecules to an activated receptor. As well as the SH2 domain, Src-family kinases possess another binding domain known as SH3 or Src homology 3. This domain, which is also found in other proteins, binds to proline-rich regions in diverse proteins and can thus recruit these proteins into the signaling pathway, as we will see later. Src-family kinases are usually anchored to the cell membrane by a lipid moiety attached to their amino-terminal region. They are distributed over the inner surface of the cell membrane; during cell activation they become localized to sites of receptor signaling by binding to phosphotyrosine via their SH2 domains.

As a signaling mechanism, phosphorylation also has the advantage that it is easily and rapidly reversible by protein phosphatases that specifically remove the phosphate groups added by the protein kinases. It is crucial that components of signaling pathways can be readily returned to their unstimulated state; not only does this make the signaling pathway ready to receive another signal but it sets a limit on the time that any individual signal is active, preventing cellular responses from running out of control. It is not surprising, therefore, that the signaling pathways that link the cell surface to changes in gene expression use protein phosphorylation and dephosphorylation to regulate the activity of many of their components.

6-4. Intracellular signaling components recruited to activated receptors transmit the signal onward from the membrane and amplify it

Several classes of protein are typically recruited to the activated receptors and participate in signal propagation. The enzyme phospholipase C-γ (PLC-γ) contains two SH2 domains through which it can bind to phosphotyrosine; it is thus recruited to the site of receptor-associated tyrosine kinase activity at the plasma membrane. PLC-γ has a crucial role in propagating the signal onward from the membrane and in amplifying it. Phosphorylation of a tyrosine residue in PLC-γ activates the enzyme, which then cleaves molecules of the membrane phospholipid phosphatidylinositol bisphosphate (PIP₂) into two parts, inositol trisphosphate (IP₃) and diacylglycerol (DAG) (Fig. 6.4). As one molecule of PLC-γ can generate many molecules of DAG and IP₃, this and similar enzymatic steps serve to amplify and sustain the signal. Production of DAG and IP₃ by activated PLC-γ is a common step in pathways from many types of receptor.

Figure 6.4

The enzyme phospholipase C-γ cleaves inositol phospholipids to generate two important signaling molecules. Phosphatidylinositol bisphosphate (PIP₂) is a component of the inner leaflet of the plasma membrane. When phospholipase C-γ (PLC-γ) (more...)

Interaction of IP₃ with its receptors in the endoplasmic reticulum causes the release of Ca²⁺ into the cytosol from this intracellular storage site, immediately raising intracellular free Ca²⁺ levels severalfold. Depletion of the endoplasmic reticulum calcium stores triggers the opening of calcium channels in the plasma membrane that let more Ca²⁺ into the cell, thus sustaining the signal (see Fig. 6.4). Increased intracellular free Ca²⁺ leads to the activation of the Ca²⁺- binding protein calmodulin, which in turn binds to and regulates the activity of several other proteins and enzymes in the cell, transmitting the signal onward along pathways that eventually converge on the nucleus. One protein that is regulated by the calcium pathway is the nuclear factor of activated T cells (NFAT), a transcription factor we will discuss further in Section 6-11.

The other product of PIP₂ cleavage is DAG, which remains associated with the inner surface of the plasma membrane. DAG helps to activate members of the protein kinase C (PKC) family (see Fig. 6.4). These are serine/threonine protein kinases that are thought to initiate several signaling pathways leading to the nucleus. Some isoforms of PKC are further activated by the Ca²⁺ released by IP₃ action. Thus, the two products of the cleavage of PIP₂ reinforce each other in activating PKC as well as having their own independent effects.

Many of the cellular processes that are activated in lymphocytes when antigen binds to its receptor are common to many cell types. For example, resting lymphocytes proliferate when exposed to antigen, whereas other cell types proliferate in response to particular growth factors; what differs in each case is the receptor that initiates the common response pathway in the different cell types. To link these different receptors to common intracellular signaling components, specialized adaptor proteins are needed.

In lymphocytes, the adaptor proteins that bind to the antigen receptors contain two or more domains, for example, SH2 and SH3 domains, that mediate protein-protein interactions. These proteins do not have kinase activity themselves and their function in general is to recruit other molecules to the activated receptor. Such a protein can bind to a phosphotyrosine residue via its SH2 domain and to other proteins containing proline-rich motifs via its SH3 domains (Fig. 6.5). Binding to an adaptor protein positions these other proteins at or near the cell membrane, where they can in turn be phosphorylated and activated by the tyrosine kinases associated with the receptor. One important family of proteins whose members are bound by adaptors and activated during signaling are the guanine-nucleotide exchange factors (GEFs), which, as we will see in the next section, pass the signal on to another common central component of many signaling pathways, the small G proteins.

Figure 6.5

Signals are propagated from the receptor through adaptor proteins, which recruit other signaling proteins to the receptor. Adaptor proteins are specialized signaling molecules that usually have no enzymatic activity themselves. Instead, they allow other molecules (more...)

6-5. Small G proteins activate a protein kinase cascade that transmits the signal to the nucleus

Small GTP-binding proteins or small G proteins are another class of protein that serves to propagate signals from tyrosine kinase-associated receptors. The family of small single-chain GTP-binding proteins is distinct from the heterotrimeric G proteins that associate with seven-span transmembrane receptors such as the anaphylotoxin or chemokine receptors (see Section 6-16). The best-known small G protein is Ras. Like the Src-family kinases, Ras was discovered through its effects on cell growth. A gene encoding a mutated form of Ras was found in various animal retroviruses that cause tumors, and the corresponding cellular Ras gene (c-Ras) was subsequently found to be mutated in many different human tumors. The frequent discovery of mutant c-Ras in tumors indicated that the normal Ras gene had a critical role in the control of cell growth and focused attention on the physiological role of Ras. This protein has been highly conserved throughout evolution. Ras proteins are found in all eukaryotic cells and are activated in response to many different cell-activating ligands.

Small G proteins such as Ras exist in two states, depending on whether they are bound to GTP or to GDP. The GTP-bound form of Ras is active, but it can be converted into an inactive GDP-bound form by an intrinsic Ras-GTPase activity, which removes a phosphate group from the bound GTP. This reaction is accelerated by regulatory cofactors, and small G proteins do not normally stay permanently activated; instead they eventually turn themselves off. Thus the small G proteins are usually found in the inactive GDP-containing state, and they are activated only transiently in response to activating ligands. However, mutation at a single residue can lock them in the active state, causing them to become oncogenic.

The activation of small G proteins is mediated by guanine-nucleotide exchange factors (GEFs), which exchange GDP for GTP (Fig. 6.6). In lymphocytes, Ras and other small G proteins are recruited to the receptor site by adaptor proteins and are activated by GEFs bound to these adaptor proteins. Thus, G proteins can act as molecular switches, becoming switched on only when the cell-surface receptor is activated.

Figure 6.6

Small G proteins are switched from inactive to active states by guanine-nucleotide exchange factors. Ras is a small GTP-binding protein with intrinsic GTPase activity. In its GTP-bound state, it is active (first panel), whereas in the GDP-bound state (more...)

Once activated, small G proteins activate, among other things, a cascade of protein kinases known as the mitogen-activated protein kinase (MAP kinase) cascade. This kinase cascade is found in all multicellular animals and is responsible for many of the effects of activating ligands. The MAP kinase cascade leads directly to the phosphorylation and activation of transcription factors in the nucleus. In particular, the AP-1 family of transcription factors, which are heterodimers of the Fos and Jun proteins, the products of the oncogenes fos and jun, are activated through MAP kinase cascades. We will discuss these activation pathways in more detail in the next part of the chapter, where the structures of the lymphocyte antigen-receptor complexes are described and we look at the particular signals generated by the antigen receptors and the co-receptors that cluster with them.

Summary

Lymphocyte antigen receptors signal for cell activation using signal transduction mechanisms common to many intracellular signaling pathways. On ligand binding, antigen receptor clustering leads to the activation of receptorassociated protein tyrosine kinases at the cytoplasmic face of the plasma membrane. These initiate intracellular signaling by phosphorylating tyrosine residues in the clustered receptor tails. The phosphorylated tyrosines act as binding sites for additional kinases and other signaling molecules that amplify the signal and transmit it onward. The enzyme phospholipase C-γ is recruited in this way and initiates two major pathways of intracellular signaling that are common to many other receptors. Cleavage of the membrane phospholipid PIP₂ by this enzyme produces the diffusible messenger inositol trisphosphate (IP₃) and membrane-bound diacylglycerol (DAG). IP₃ action leads to a sharp increase in the level of intracellular free Ca²⁺, which activates various calcium-dependent enzymes. Together with Ca²⁺, DAG initiates a second signaling pathway by activating members of the protein kinase C family. A third pathway involves small G proteins, proteins with GTPase activity that are activated by binding GTP, but then hydrolyze the GTP to GDP and become inactive. Small G proteins are recruited to the signaling pathway and activated by guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP. GEFs and other signaling molecules are linked to the activated receptors by adaptor proteins that bind to phosphorylated tyrosines through one protein domain, the SH2 domain, and to other signaling molecules through other domains including SH3. All these signaling pathways eventually converge on the nucleus to alter patterns of gene transcription.