Principles of Signaling

Key Points

  • Cells of the immune system sense environmental stimuli via receptors, which may be expressed on the cell surface or may be intra-cellular.

  • Interaction of ligands with receptors initiates signaling cascades, which relay the extra-cellular stimuli within the cell and alter cellular function.

  • Signaling pathways typically involve phosphorylation (by kinases) and dephosphorylation (by phosphatases) of molecules.

  • Signaling eventually leads to cellular responses such as changes in growth, activation, proliferation, and differentiation.


Immune cells respond to a vast variety of stimuli to carry out their role in maintaining the immune system. Physiologic and innocuous and foreign or dangerous signals must be recognized and communicated within the cell, where they culminate into cellular responses such as changes in shape, motility, growth, activation, differentiation, or production of effector molecules. Distinct cascades of interacting molecules connect the perceived stimuli and relay information into the cytosol and/or nucleus to initiate these effector functions either directly or through initiation of gene transcription and protein translation programs. Signaling pathways can be categorized based on the mechanisms by which environmental stimuli are sensed, such as cell surface receptor-mediated interactions or intra-cellular detection of lipid-soluble molecules. Receptor-mediated signaling can further be classified by the presence or absence of enzymatic activity. This chapter focuses on fundamental concepts of signaling based on these groups of receptors and their intra-cellular signaling pathways.

Receptors With Enzymatic Activity

Many ligands are water soluble and therefore impermeable through the lipid bilayer of the plasma membrane; they interact with cognate receptors on the cell surface. These ligands include antigens, immune complexes, chemokines, cytokines, and microbial components. Interactions of ligands with their receptors initiate downstream catalytic activity and recruitment of similar signaling molecules or adaptor molecules. Receptors with enzymatic activity comprise extra-cellular ligand-binding domains, membrane-spanning domains, and intra-cellular signaling domains. Intrinsic enzymatic activity within the receptor includes kinase, phosphatase, or guanylate cyclase activity.

The receptor tyrosine kinase (RTK) family is a common class of these receptors. The human RTK family consists of 20 subfamilies that detect ligands such as stem cell factor (SCF), insulin, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), colony-stimulating factor (CSF), and fibroblast growth factor (FGF). These receptors are generally inactive until bound by their ligands, which induce clustering and dimerization of the receptors and autophosphorylation of their kinase domains ( Fig. 18.1A ). Upon activation, other kinases or cytoplasmic molecules are recruited for activation and transmission of signals downstream. In addition to receptor binding, ligands, along with accessory molecules, may also mediate receptor dimerization. FGF, for example, partners with heparan sulfate proteoglycans to cross-link and dimerize FGF receptors (FGFR). Some receptors may dimerize even in the absence of ligands, such as the disulfide-linked insulin receptor and the insulin-like growth factor (IGF)-1 receptors. Ligand binding induces structural changes to activate these receptors and signaling by diverse mechanisms.

Fig. 18.1

(A) Receptor tyrosine kinase c-kit, the receptor for stem cell factor (SCF) is depicted in the inactive form as monomers (left) . Upon binding to its ligand, the receptor dimerizes, and the kinase domains phosphorylate each other (right) . (B) Schematic depicting the transforming growth factor (TGF)-β–TGFβR signaling pathway. P, Phosphate.

Phosphorylation occurs at tyrosine, serine, or threonine residues. The phosphotyrosine residue is recognized by proteins containing Src homology (SH)2 and phosphotyrosine-binding (PTB) domains. These proteins may be kinases or phosphatases or lack enzymatic activity and act as intermediates known as adaptor proteins . SH2 domain proteins include phospholipase Cγ (PLCγ), phosphatidyl inositol-3-OH kinase (PI3K), and SHP phosphatases. Grb-2 and IRS are examples of adaptor proteins, of which Grb-2 recruits the guanine nucleotide exchange factor (GEF) SOS. SOS activates the small G protein Ras to activate the mitogen-activated protein kinase (MAPK) pathway.

Transforming growth factor (TGF)-β is an anti-inflammatory cytokine, which binds to receptors similar to the RTK family and regulates a variety of cellular processes. The TGF-β ligand superfamily includes bone morphogenetic proteins, growth and differentiation factors (GDFs), activin, and TGF-β1, 2, and 3. TGF-β binding to a type II receptor that bears a serine threonine kinase mediates phosphorylation of a type I receptor to form a heterotrimeric complex with the ligand. This complex recruits and phosphorylates the intra-cellular SMAD proteins. The SMAD proteins include the receptor-activated (r)-Smads 1, 2, 3, 5, and 9. Once phosphorylated, the r-Smads 2 and 3 oligomerize with a common mediator, Smad4, and translocate to the nucleus to regulate gene transcription (see Fig. 18.1B ).

Some receptors have phosphatase activity and are known as protein tyrosine phosphatases (PTP). For example, the Src family kinases contain an activating and an inhibitory tyrosine residue. C-terminal Src kinase (Csk) phosphorylates the inhibitory tyrosine, whereas the transmembrane tyrosine phosphatase CD45 removes this phosphate, which is required for initiation of signaling in lymphocytes.

Receptors That Recruit Molecules With Enzymatic Activity

Immunoreceptors: T Cell Receptor, B Cell Receptor, and FcRs

Immune cells express antigen recognition receptors that are generated and selected for in a complex fashion. , T cells and NKT cells express T cell receptor (TCR) and recognize peptides or lipids associated with the major histocompatibility complex (MHC), or CD1d, respectively. B cells, through the B cell receptor (BCR), recognize soluble antigens. Lymphoid and myeloid cells, such as NK cells and macrophages, express receptors of the FcR family, which cause activation upon detection of antibodies in complex with antigen. These receptors are heteromultimeric complexes, with a subunit of one or two chains that recognize the ligand, co-receptors (CD4, CD8, or CD19), and associated or adaptor molecules that contain from one to six immunoreceptor tyrosine-based activation motif (ITAM) domains ( Fig. 18.2 ).

Fig. 18.2

Schematic depicting signaling pathways through cytokine receptors, G protein–coupled receptors (GPCRs), and immune receptors. AC, Adenyl cyclase; AP-1, activator protein-1; GADS, growth factor receptor-bound protein-2-related adaptor protein-2; ITAM, immunoreceptor tyrosine activation motif; JAK, Janus kinase; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells; PI3K, phosphatidyl inositol-3-OH kinase; PIP 3 , phosphatidylinositol-3,4,5-triphosphate; PKA, protein kinase A; PLC, phospholipase C; SLAP, Src-like adaptor protein; STAT, signal transducer and activator of transcription; TNFR, tumor necrosis factor receptor; TRAF, TNFR-associated factor; TRADD, TNFR-associated death domain.

Antigen recognition by the immune receptor and co-receptor triggers the recruitment and activation of membrane proximal early kinases, including Src, Syk, and Tec, which phosphorylate tyrosines of the ITAM domains in the chains associated with the immune receptors. The Src family phosphotyrosine kinases (PTK) comprise eight kinases (Fgr, Fyn, Src, Yes, Blk, Hck, Lck, Lyn), which are recruited through their SH2 domain. The Tec family consists of five different kinases (Bmx, Btk, Itk, Tec, and Txk/Rlk), which translocate to the cell membrane through their pleckstrin homology (PH) domain to bind PIP 3 . Alternatively, they are recruited via their SH2 domains, which recognize ITAM motifs on adaptor proteins (e.g., BLNK in B cells, or SLP-76 and LAT in T cells). The principal downstream target of Tec kinases is PLCγ. , Their function is regulated by the kinase Csk and the protein tyrosine phosphatase CD45.

ITAM phosphorylation leads to recruitment and activation of Syk family kinases recruited through SH2 domains. BCRs recruit Syk, whereas ZAP-70 binds to phospho-ITAMs of CD3ζ within the TCR. Both kinases are implicated in the signaling of Fc receptors because Syk associates with their common gamma chain, although some incorporate CD3ζ to recruit ZAP-70. These then undergo autophosphorylation to create sites for interaction with SH2 proteins.

Co-stimulatory Receptors

T cell activation requires two signals, one mediated through the T cell receptor and a second signal called co-stimulation . This signal is provided by a family of receptor-ligand molecules that mediate interactions between T cells and antigen-presenting cells. The B7/CD28 is one of the most well-studied pathways of co-stimulation. B7-1 and B7-2 are expressed by APCs and bind CD28 on T cells. This interaction triggers downstream signaling to amplify the primary signal and initiate effector responses. CD28 is phosphorylated by Lck and activates the PLCγ and PI3K/Akt pathways. An important consequence of this signaling pathway is to enhance mRNA stability of IL-2, which is responsible for the dramatic increase in IL-2 secretion. Cytotoxic T lymphocyte antigen (CTLA)4 is closely related to CD28 in structure, can bind to B7 molecules but with much higher avidity, and curbs the activation and proliferation of T cells. In addition to the B7-CD28 pairing, other co-stimulatory molecules include the inducible co-stimulator (ICOS), which is induced on activated T cells and interacts with B7-H2/ICOSL on activated dendritic cells, monocytes, and B cells. Another class of co-stimulatory molecules is the signaling lymphocyte activation molecule (SLAM) family, a subtype of the immunoglobulin superfamily consisting of nine transmembrane proteins. SLAMs are important for multiple cell types, including T, B, and NK cells, and mediate their effects via homophilic and heterophilic interactions. The SLAM proteins bear a tyrosine-based switch motif through which they bind SH2-bearing proteins SLAM-associated protein (SAP) and EAT2 with high affinity.

Once activated, T cells express other co-stimulatory molecules, such as CD40L and the programmed cell death (PD)1 receptor. CD40L interacts with CD40 on APCs, and this induces signals, which play an important role in effector functions, such as activation of B cells, to produce antibodies. A co-stimulatory inhibitory pathway involves PD1 on T cells binding to B7-H1/PDL1 and B7-DC/PDL2 expressed on APCs. PD1 belongs to the immunoglobulin superfamily, and its intra-cellular domain bears an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The tyrosine in the ITSM binds to the phosphatases SHP1 and SHP2, which act to inhibit the PI3K/Akt pathway. PD1 signaling inhibits induction of the antiapoptotic molecule BcL-xL and transcription factors such as T-bet, GATA-3, and Eomes, which are important for T cell differentiation.

Thus, co-stimulatory signals not only activate cells to promote proliferation and effector functions required in the immune response but also are inhibitory to curb excessive activation and are important for tolerance induction.

Cytokine Receptors

Cytokines are soluble peptide regulators with profound effects on immune homeostasis and autoinflammatory disease. Cytokine receptors are organized into four classes on the basis of their domain and structure:

  • 1.

    Class I receptors are characterized by a WSXWS motif and can be subdivided on the basis of structural homology or the use of shared signaling subunits. Examples include receptors for IL-2, IL-6, IL-7, IL-15, and for hormones such as erythropoietin and prolactin.

  • 2.

    Class II receptors include those for type I and II interferon (IFN), IL-10, IL-20, IL-22, IL-26, IL-28, and IL-29. IFNs are glycoproteins commonly generated as a downstream effect of pathogen detection. They are divided into three classes on the basis of the receptors that they activate. Type I IFNs bind the IFNA receptor (IFNAR); this group includes IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω. IFN-γ is the sole member of type II in humans, and it binds IFNGR. Type III IFNs activate a receptor complex consisting of IL-10R2 and IFNLR1; this group includes IL-29, IL-28A, and IL-28B. Activation stimulates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal cascades and transcriptional changes made in cooperation with interferon regulatory factor (IRF) proteins. The expression of so-called interferon signature genes (ISG) promotes anti-viral activities, increased MHC presentation, and apoptosis.

  • 3.

    TNF receptors share structures with some noncytokine receptors, such as CD95. The TNF superfamily consists of 19 ligands and 29 receptors, which play varied roles in inflammation, apoptosis, and proliferation. TNF and lymphotoxin-α (also known as TNFβ) were the first two members identified and share approximately 50% homology in their protein sequence. Other members of this superfamily include lymphotoxin-β, CD40L, FasL, CD30L, 4-1BBL, CD27L, OX40L, TNF-related apoptosis-inducing ligand (TRAIL), lymphotoxin-like ligand competitive with gD-1 HSV for HVEM expressed on T cells (LIGHT), receptor activator of NF-κB ligand (RANKL), TNF-related weak inducer of apoptosis (TWEAK), a proliferation-inducing ligand (APRIL), B cell–activating factor (BAFF), vascular endothelial cell-growth inhibitor (VEGI), ectodysplasin A (EDA)-A1, EDA-A2, and glucocorticoid-induced TNF receptor (TNFR) family ligand (GITRL). TNF binds to two distinct receptors: TNFR1 and TNFR2 ( Fig. 18.3 ). Receptors of the TNF superfamily can be categorized based on the presence or absence of an intra-cellular death domain (DD), of which the D-containing receptors are ubiquitous in expression. TNFR1 contains a DD and is expressed in virtually all cell types, whereas TNFR2 is expressed mainly on immune cells, endothelial cells, and nerve cells. TNF is expressed both in soluble form and as a transmembrane protein on the cell surface, whereas lymphotoxin-α is only expressed as soluble protein. TNF signaling induces several signaling pathways, including activation of NF-κB, MAPK, and apoptosis.

    Fig. 18.3

    Tumor necrosis factor receptor 1 (TNFR1) and TNFR2 signaling pathways. AP, Activator protein; BCL, B cell lymphoma; cIAPs, cellular inhibitors of apoptosis; COX, cyclooxygenase; FADD, Fas-associated protein with death domain; FLIP, FLICE-like inhibitory protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; IAP, inhibitor of apoptosis; ICAM, intercellular adhesion molecule; IKK, IκB (inhibitor of κB) kinase; iNOS, inducible nitric oxide synthetase; MAPK, mitogen-activated protein kinases; MCP, monocyte chemotactic protein; MMP, matrix metalloproteinase; NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; RIP, receptor-interacting serine/threonine-protein kinase 1; TANK, TRAF family member-associated NF-κB activator; TRADD, tumor necrosis factor receptor type 1–associated death domain; TRAP, TNF receptor-associated protein; VCAM, vascular cell adhesion protein; XIAP, X-linked inhibitor of apoptosis protein.

  • 4.

    IL-1 receptors can be considered analogous to Toll-like receptors (TLRs).

These receptor complexes present two or more single-pass transmembrane subunits that include domains that recruit signaling proteins upon activation and subunits with extra-cellular binding domains. Whereas class I and II cytokine receptors use the JAK-STAT signaling pathway, TNFRs use adaptor molecules called TNFR-associated factors (TRAFs) to recruit complexes with different functions.

Adhesion Molecules

A variety of cellular functions, including lymphocyte activation, migration, and cell-cell interaction, involve adhesion molecules. These include selectins, integrins, and immunoglobulin superfamily molecules. L-selectin (CD62L) is important for lymphocyte homing to lymphoid tissues. Activated lymphocytes downregulate L-selectin and upregulate other adhesion/migratory molecules, such as CD44, a transmembrane protein, which recognizes hyaluronic acid and is critical for migration into peripheral sites of inflammation. CD44 lacks intrinsic kinase activity, but the cytoplasmic domain associates with Src family kinases Lck and Fyn. In addition, the cytoplasmic tail binds to the phosphorylated ezrin/radixin/moesin (pERM) proteins, which cross-link actin cytoskeleton proteins to CD44. CD44 signaling also activates PI3K/Akt cell survival pathways.

Integrins are heterodimeric cell surface proteins that provide interactions with other cells and the extra-cellular matrix, allowing for migration to effector sites. Various chemokines can activate integrins to mediate migration into lymphoid or nonlymphoid tissues. T lymphocyte integrins include leukocyte function–associated antigen (LFA)-1, LPAM-1, and very late activation antigen (VLA)-4, which bind immunoglobulin superfamily members ICAM-1, MAdCAM-1, and VCAM-1, respectively. Integrin activation results in inside-out signaling, which regulates the affinity of the integrin receptor to the extra-cellular ligands. Elements of this signaling pathway include the small GTPase RAP1 and its GEFs talin and kindlin3.

Seven-Transmembrane Domain Receptors

G Protein–Coupled Receptors

G protein–coupled receptors (GPCRs) are a large family of approximately 350 members that bind a variety of ligands, including hormones, lipids, chemokines, and leukotrienes. They are seven-transmembrane domain-bearing proteins that associate with an intra-cellular trimeric G protein (α, β, or γ) and are organized into five families: rhodopsin, secretin, glutamate, adhesion, and Frizzled/Taste2 ( Table 18.1 ). Ligand binding promotes the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) and dissociation of the Gα and β subunits from the receptor subunit.

TABLE 18.1

Examples of G Protein–Coupled Receptors (GPCR) and Their Ligands

GPCR Superfamily GPCR Family GPCR Ligands
Rhodopsin Adenosine receptor A 2A R Adenosine
Chemokine receptor CXCR4 SDF1
Bradykinin receptor B 2 R Bradykinin
Anaphylatoxin receptor C5aR C5a
S1P receptor S1P1 S1P
Protease-activated receptor PAR-1 Thrombin
PAR-2 Trypsin
Prostaglandin receptor EP 2 /EP 4 Prostaglandin E2
Adhesion CD97 CD55
Secretin GCG-R Glucagon
Glutamate Kainate GluR Glutamate
Frizzled/Taste2 FZ5 Wnt5a

Based on their α-subunits, G proteins are divided into four subfamilies: Gαs, Gαi/o, Gαq/11, and Gα12/13. Each Gα has specific targets: Gαs and Gαi/o activate or repress adenyl cyclase (AC), regulating cyclic adenosine monophosphate (cAMP) levels to impact several ion channels and the activation of protein kinase A (PKA), whereas Gαq/11 activates phospholipase C (PLC)-β to generate IP 3 and diacylglycerol (DAG) from PIP 2 , ultimately leading to Ca 2+ and protein kinase C (PKC) pathway activation. The targets for Gα12/13 are three RhoGEFs that activate the small GTPase Rho, critical for cytoskeleton regulation via the stress-activated MAPK pathway. Alternatively, the Gβγ-complexes regulate several ion channels, specific isoforms of AC, PLC, and PI3K (see Fig. 18.2 ). Gβγ dimers activate the Rho family G proteins to regulate actin filament reorganization and therefore induce cytoskeletal changes and impact cellular trafficking. These changes are important in response to chemokines, anaphylatoxins, or histamine. Sphingosine-1-phosphate (S1P) is a lipid-signaling molecule present in blood and lymph. Its receptor is abundantly expressed on endothelial cells and is important for lymphocytes exiting from the thymus and lymph nodes.

Wingless Type Signaling Pathways

The wingless type (Wnt) signaling pathway is a complex signaling pathway that, depending on the nature of the ligands and downstream events, can be divided into two classes: the canonical and the noncanonical pathways. The canonical pathway includes Wnt1 class ligands (Wnt2, Wnt3, Wnt3a, and Wnt8a), which bind to Frizzled (Frz) receptors and their co-receptors, LRP5 and LRP6. When activated, Frz and LRP recruit and inhibit the cytoplasmic APC/Axin destruction complex, integrated by protein kinases such as casein kinase (CK)-1 and glycogen synthase kinase (GSK)-3b, the tumor suppressor adenomatous polyposis coli (APC) protein, and the scaffolding protein Axin, to stabilize β-catenin. The canonical Wnt pathway is mainly involved in cell proliferation and differentiation.

Noncanonical Wnt signaling is initiated by the Wnt5a types (Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, and Wnt11), which bind to Frz receptors, activate Dishevelled (Dvl), and, depending on the phenotypic response, are classified as Wnt/planar cell polarity (PCP) or Wnt/Ca 2+ pathways. , The Wnt/Ca 2+ pathway activates the Phospholipase C (PLC) pathway, which leads to the release of calcium and the activation of protein kinase C, CaMKII, and calcineurin, as well as the transcription factor nuclear factor associated with T cells (NFAT), to regulate cytoskeletal rearrangements, cell adhesion, and migration. In the PCP pathway, Wnt ligands are recognized by Frz/retinoic acid-related orphan receptor (ROR)/RTK complexes. This pathway activates Rho and Rac, leading to cytoskeletal rearrangements and cell motility through the activation of Rho‐associated kinase (ROCK) and the c‐Jun N‐terminal kinase (JNK) signaling pathway.

Innate Receptor Signaling

TLRs and nucleotide-binding oligomerization-like receptors (NLRs) constitute the major pathogen sensors of innate immunity and work in a cell autonomous fashion to initiate anti-microbial responses, including inflammation and apoptosis ( Fig. 18.4 ). The major downstream effect of the cascades initiated by pathogen-associated molecular pattern (PAMP) recognition is IRF transcriptional activation of IFN genes that mediate inflammation and tissue repair. PAMPs are detected in endosomes and on the surface of cells by a variety of TLRs. These receptors have leucine-rich repeat domains to detect ligands, a single-pass membrane domain, and an intra-cellular Toll–interleukin-1 receptor (TIR) motif for downstream signaling. Their activities are most relevant in macrophages, monocytes, and dendritic cells.

May 20, 2021 | Posted by in RHEUMATOLOGY | Comments Off on Principles of Signaling

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