Most of the receptors that initiate signaling responses are integral membrane proteins present on the plasma membrane, where their extracellular domains recognize soluble secreted ligands or structures that are attached to the plasma membrane of a neighboring cell or to the extracellular matrix. Another category of receptors, nuclear receptors, are transcription fac tors that are activated by lipid-soluble ligands that can cross the plasma membrane.
The initiation of signaling from a cell surface receptor may require ligand-induced clustering of receptor proteins, called cross-linking, or may involve a conformational alteration of the receptor induced by its association with ligand. Both mechanisms of signal initiation typically result in the creation of a novel shape in the cytosolic portion of the receptor that pro motes the recruitment of and/or interactions with other signaling molecules.
A common early event in signal transduction is the enzymatic addition of a phosphate residue on the side chain of an amino acid in the cytosolic portion of a receptor or in other proteins that are involved in signaling; sometimes a phosphate residue is added to a lipid on the inner leaflet of the plasma membrane. The enzymes that add phosphate groups onto amino acid side chains are called protein kinases. Many of the initiating events in lymphocyte signaling depend on protein kinases that phosphorylate specific tyrosine residues; these enzymes are therefore called protein tyrosine kinases. Other protein kinases that are involved in distinct signaling pathways are serine/threonine kinases, which phosphorylate serine or threonine residues. The enzymes activated downstream of signaling receptors that phosphorylate lipid substrates are known as lipid kinases. For most known phosphorylation events, there are also specific phosphatases—enzymes that can remove phosphate residues and thus modulate signaling. These phosphatases play important, usually inhibitory, roles in signal transduction.
Phosphorylation of proteins is not the only posttranslational modification that drives signal transduction. Many other modifications can facilitate signaling events. A type of modification that we will describe later in this chapter is the covalent addition of ubiquitin molecules that either target proteins for degradation or drive signal transduction in many cells, including lymphocytes. Many important signaling proteins are modified by the addition of lipids that may help localize these proteins to a specialized region of the plasma membrane in order for them to efficiently interact with other signaling molecules that are also targeted to this membrane microdomain. Some transcription factors are functionally modified by acetylation, and the N-terminal tails of histones can be acetylated and methylated in order to modulate gene expression, DNA replication, and DNA recombination events.
Cellular receptors are grouped into several categories based on the signaling mechanisms they use and the intracellular biochemical pathways they activate (Fig.1):
• Some receptors have no intrinsic enzymatic activity and use nonreceptor tyrosine kinases for initiating signals. The nonreceptor tyrosine kinase is an intracellular enzyme that is separate from the receptor and is activated by ligand binding to the receptor. These kinases participate in cellular responses by phosphorylating specific motifs on the receptor or on other proteins associated with the receptor (see Fig. 2). Nonreceptor kinases are used by members of the immune receptor family, whose members include antigen receptors on lymphocytes, activating and inhibitory receptors on NK cells, and receptors for the Fc portions of antibodies on various cell types including many myeloid cells. In addition to the immune receptor family, some cytokine receptors, dis cussed later in this chapter, use nonreceptor tyrosine kinases. Integrins, key adhesion receptors in the immune system, also signal in part by activating nonreceptor tyrosine kinases.
• Receptor tyrosine kinases (RTKs) are integral membrane proteins that contain an intrinsic tyrosine kinase domain (or domains) located in the cytoplasmic tail that is activated when the receptors are cross-linked by multivalent extracellular ligands. This category of receptors is important in the responses of many cell types, but this category does not play a major role in lymphocyte activation. An example of an RTK relevant to hematopoiesis is the c-KIT protein. Other examples include the insulin receptor, the epidermal growth factor receptor, and the platelet-derived growth factor receptor. A smaller number of receptors have cytosolic kinase domains that phosphorylate target proteins on serine or threonine residues (and not on tyrosine residues). Two examples will be discussed later in this chapter when we consider TGF-β signaling.
• Nuclear receptors are typically located within, or migrate into, the nucleus, where they function as transcription fac tors. One category of nuclear receptors is made up of proteins that reside in the cytosol at steady state and, after they bind their ligands, migrate into the nucleus and regulate specific transcription events. A distinct category of nuclear receptors resides in the nucleus at steady state. The binding of a lipid-soluble ligand to its nuclear receptor results in the ability of the receptor to stimulate or repress gene transcription. Nuclear hormone receptors, such as the vitamin D receptor and the glucocorticoid receptor, can influence maturation and activation of immune cells and modulate cytokine gene expression.
• G protein–coupled receptors (GPCRs) are a large family of membrane receptors that function by activating associated GTP-binding proteins (G proteins). These receptors are poly peptides that traverse the plasma membrane seven times, because of which they are sometimes called serpentine receptors or seven-transmembrane receptors. A conformational change induced by the binding of ligand to GPCRs permits the activation of an associated heterotrimeric G protein by the exchange of bound GDP with GTP. The activated G protein initiates downstream signaling events. Examples of GPCRs that are relevant to immunity and inflammation include receptors for leukotrienes, prostaglandins, histamine, complement fragments C3a and C5a, bacterial formyl peptides, sphingosine-1-phosphate, and all chemokines. Different types of G proteins linked to distinct GPCRs may activate or inhibit different downstream effectors. Two major enzymes that GPCRs activate are adenylate cyclase, which converts ATP to the effector molecule cAMP, capable of activating numerous cellular responses, and phospholipase C, which also triggers multiple signaling events as discussed later.
• Other classes of receptors have long been known to be important in embryonic development and in certain mature tissues, and their functions in the immune system have begun to emerge more recently. Receptor proteins of the NOTCH family are involved in development in a wide range of species. The association of specific ligands with receptors of this family leads to proteolytic cleavage of the receptor and the nuclear translocation of the cleaved cytoplasmic domain (intracellular NOTCH), which functions as a component of a transcription complex. NOTCH proteins contribute to cell fate determination during lymphocyte development. A group of ligands called WNT proteins can influence lymphopoiesis. (The names of many proteins involved in signaling are often based on how they were discovered and do not reflect their functions, so we will use generally accepted abbreviations and not list the full names.) Signaling through transmembrane receptors for these proteins can increase the levels of β-catenin, which can enter the nucleus and activate transcription factors that contribute to B- and T-cell development. Numerous other signaling receptors and pathways first discovered in nonimmune cell populations are now being studied in the context of lymphocyte biology. We will not attempt to comprehensively consider all of these pathways in this chapter.
Fig1. Major categories of signaling receptors in the immune system. Depicted here are a receptor that uses a nonreceptor tyrosine kinase; a receptor tyrosine kinase; a nuclear receptor that binds its ligand and can then influence transcription; a seven-transmembrane G protein–coupled receptor (GPCR); and NOTCH, which recognizes a ligand on a distinct cell and is cleaved, yielding an intracellular fragment (IC Notch) that can enter the nucleus and influence transcription of specific target genes. ATP, Adenosine triphosphate; cAMP, cyclic adenosine monophosphate.
Fig2. Signaling from the cell surface involves cytosolic and nuclear phases. A generic receptor that activates a nonreceptor tyrosine kinase after it binds ligand is shown. In the cytosolic signaling phase, the nonreceptor kinase phosphorylates a key tyrosine residue on the cytoplasmic tail of the receptor, as a result of which the phosphotyrosine-containing receptor tail is able to recruit a downstream enzyme that is activated once it is recruited. In the cytosolic phase, this activated downstream enzyme post-translationally modifies a specific transcription factor that is located in the cytoplasm. In the nuclear phase, this modified transcription factor enters the nucleus and induces the expression of target genes that have a binding site in the promoter or in some other regulatory region that can bind to this modified transcription factor and facilitate transcription. In this simplified example, the cytosolic phase has only a single enzymatic event, and only one transcription factor is activated, but most signaling pathways involve multiple enzymatic steps and may activate more than one transcription factor.
Modular Signaling Proteins and Adaptors
Signaling molecules are often composed of conformationally distinct regions called modules that have specific binding or catalytic functions. The concept of modular signaling molecules has been best illustrated from the study of nonreceptor tyrosine kinases. The cellular homologue of the transforming (cancer causing) protein of the Rous sarcoma virus, called c-SRC, is the prototype for an immunologically important family of nonreceptor tyrosine kinases known as SRC family kinases. These kinases contain several distinct domains, two of which, called SRC homology 2 (SH2) and SRC homology 3 (SH3) domains, mediate binding to other signaling proteins. Src family kinases also contain a catalytic tyrosine kinase domain and an N-terminal lipid addition domain that facilitates the covalent addition of a myristic acid molecule to the protein. The myristate helps localize SRC family kinases to the plasma mem brane. The modular structures of the SRC family kinases, as well as two other families of tyrosine kinases discussed later that are important in the immune system, are depicted in Fig. 3.
Fig3. The modular structure of tyrosine kinases that influence lymphocyte activation. Modules include SH2 domains that bind specific phosphotyrosine-containing polypeptides, SH3 domains that recognize proline-rich stretches in polypeptides, PH domains that recognize PIP3 or other phosphatidylinositol-derived lipids, and TEC homology domains found in tyrosine kinases of the TEC family. Tyrosine kinase families depicted are the SRC family kinases, which include c-SRC, LYN, FYN, and LCK; the SYK family kinases, which include SYK and ZAP70; and the TEC family kinases, which include TEC, BTK, and ITK. The unique N-terminal domain of SRC-family kinases includes a myristylation site shown her attached to myristic acid. K, Kinase domain; P, proline-rich peptide; PH, pleckstrin homology; SH, src homology; T, Tec homology domain; U, unique domain.
SH2 domains are composed of about 100 amino acids folded into a particular conformation, and they bind to phosphotyrosine-containing peptides in various proteins. In antigen receptor signaling, SRC family kinases phosphorylate tyrosine residues present in certain motifs in the cytoplasmic tails of proteins that are part of the antigen receptor complex (described later). These phosphotyrosine motifs in the complex then serve as binding sites for SH2 domains present in tyrosine kinases of the SYK family, namely SYK in B cells and ZAP70 in T cells (see Fig. 3). The recruitment of SYK or ZAP70 to an antigen receptor by means of a specific SH2 domain–phosphotyrosine interaction is a key step in antigen-induced lymphocyte activation. SH3 domains are also about 100 amino acids in length, and they help mediate protein-protein interactions by binding to proline-rich, but not phosphorylated, stretches in certain proteins. Another type of modular domain, called the pleckstrin homology (PH) domain, can recognize specific phospholipids. The PH domains in a number of signaling molecules, including the TEC family tyrosine kinase BTK, recognize phosphatidylinositol trisphosphate (PIP3), a lipid moiety on the inner leaflet of the plasma membrane.
Adaptor proteins function as molecular hubs that physically link different enzymes and promote the assembly of complexes of signaling molecules. Adaptors may be integral membrane proteins, such as LAT (Fig. 4), or they may be cytosolic proteins such as BLNK, SLP76, GRB2, and GADS. A typical adaptor may contain a few different domains that mediate distinct protein-protein interactions, such as those involving SH2 or SH3 domains (there are many more types of modular domains not mentioned here). Adaptors often contain tyrosine residues that may be phosphorylated by tyrosine kinases and serve as docking sites for SH2 domains in some signaling molecules, and the adaptors also contain proline-rich stretches that can bind SH3 domains in other signaling molecules. The amino acid residues that are close to a phosphorylated tyrosine moiety determine which specific SH2 domain–containing proteins may bind at that site. For example, a tyrosine kinase may phosphorylate a YxxM motif (where Y represents tyrosine, M represents methionine, and x refers to any amino acid) in an adaptor protein, and this will permit binding of an SH2 domain in the lipid kinase, phosphatidylinositol 3-kinase (PI3 kinase), but will not recruit other proteins that contain slightly different SH2 domains that are not specific for the phosphorylated YxxM motif. A proline-rich stretch in the same adaptor protein may bind an SH3 domain in a different tyrosine kinase. Thus, tyrosine phosphorylation of the adaptor can result in a tyrosine kinase and PI3-kinase being perched next to each other, resulting in the phosphorylation and activation of PI3-kinase. Signal transduction can therefore be visualized as a kind of social net working phenomenon. An initial signal (tyrosine phosphorylation, for instance) results in proteins being brought close to one another at designated hubs (adaptors), resulting in the activation of specific enzymes that eventually influence the nuclear localization or activity of specific downstream transcription factors or induce other cellular events, such as actin polymerization.
Fig4. Selected adaptors that participate in lymphocyte activation. On the left, LAT, an integral membrane protein that functions as an adaptor, and two cytosolic adaptors, GADS and SLP76, are shown in a nonactivated T cell. On the right, after T-cell activation, LAT is tyrosine phosphorylated and is shown to have recruited PLCγ, which simultaneously binds to the membrane phospholipid, phosphatidylinositol trisphosphate, and the GADS adaptor, both of which contain SH2 domains. A proline-rich amino acid stretch in SLP76 associates with an SH3 domain of GADS, and tyrosine-phosphorylated SLP76 recruits VAV. LAT, Linker for activation of T cells; PH, pleckstrin homology; PLC, phospholipase C; SH, src homology.
Phase Separation of Signaling Proteins
An underlying principle of signal transduction is the formation of complexes of activated signaling molecules reorganized into tiny droplets in a different phase of matter, much like the formation of a droplet of oil in water. The phase-separated proteins initiate, amplify, and propagate signals efficiently, primarily because they have been brought into close proximity and at high density. Phase separation is now recognized as a fundamental biological mechanism wherein distinct activated molecules assemble into a different phase from the neighboring constituents of a cell; this process is important in many reactions including, but not limited to, signaling and transcription. This process has also been described as being akin to “prionization,” first described in neurodegenerative diseases spread by prions, which are abnormal proteins that can propagate conformational changes to other molecules of the same protein, leading to the assembly of what is now recognized to be a phase-separated protein aggregate. There are many established examples of pathways in which phase separation of signaling proteins represents a key event in signal transduction in the immune system. These include T-cell receptor signaling, the activation of cytosolic receptors for nucleic acids and the formation of the inflammasome. Indeed, it is being increasingly appreciated that signal transduction in general depends on phase separation events that allow for the amplification and rapid propagation of signals.
