chapter 3 receptors and signaling pathway.ppt
- 1. Chapter 3 Receptors and signaling pathway 1
- 2. Common Strategies Used in Many Signaling Pathways 2
- 3. Sequence of Events Involved in Cell Signaling: Sending - Receiving - Transduction - Responding 1 2 3 When cells convert the information from one form to another Ligand (signaling molecules) binding to the receptor
- 4. • A signal transduction pathway is the molecular route by which a ligand-receptor interaction is translated into a biochemical change within the affected cell. • The upstream components of a signaling pathway are those closest to the receptor; the downstream components are those closest to the effector molecules that determine the outcome of the pathway 4
- 5. Ligand Binding • The first step necessary to the activation of a signaling pathway is that the binding of the ligand to its receptors in some way induces a physical or chemical change in the receptor itself, or in molecules associated with it. • In the case of many growth factor receptors, ligand binding induces a conformational change in the receptor that results in receptor dimerization by phosphorylation of the cytoplasmic regions of each of the receptor molecules by its dimerization partner. 5
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- 7. • Other receptors undergo conformational changes upon ligand binding that result in higher orders of receptor polymerization. • Two different types of antigen receptors exist on the surface of naïve B cells immunoglobulins M and D . • T-cell receptors similarly cluster upon antigen binding. 7
- 8. Receptor-Associated Molecules • B- and T-cell receptors have short cytoplasmic components and therefore need help from intracellular receptor-associated molecules to bring about signal transduction. • Igαβ/I(CD79/) heterodimer in B cells, and the hexameric CD3 complex in T cells are closely associated with their respective antigen receptors and are responsible for transmitting the signals initiated by ligand binding into the interior of the cell 8
- 9. • Both of these complexes have a pair of long cytoplasmic tails that contain multiple copies of the Immuno-receptor Tyrosine Activation Motif or ITAM. • ITAMs are motifs found on many signaling proteins within the immune system, which contain tyrosines that become phosphorylated following signal transduction through the associated receptor. • Phosphorylation of ITAM-tyrosine residues then allows docking of adapter molecules, thus facilitating initiation of the signaling cascade. 9
- 10. • Other molecules associated with the B- or T-cell antigen receptors may also interact with the antigen or with other molecules on the pathogen’s surface. • B cells, the CD19/CD21 complex binds to complement molecules covalently attached to the antigen. • CD4 and CD8 molecules on T cells bind to MHC molecule and aid in signal transduction; co-receptor CD28 on naïve T cells must interact with its ligands CD80 and CD86 for T-cell activation to occur. 10
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- 12. Receptor Clustering • Ligand-induced clustering of B- and T-cell receptors slows down the rates of their diffusion within the planes of their respective cell membranes, and facilitates their movement into specialized regions of the lymphocyte membrane lipid rafts. • Moving receptors and co-receptors into the lipid rafts renders them susceptible to the action of enzymes associated with those rafts. • raft -associated tyrosine kinase Lyn initiates the B-cell signaling cascade by phosphorylating the receptor- associated molecules. Lck serves a similar role in the TCR signaling cascade. 12
- 13. lipid rafts: highly ordered, detergent-insoluble, cholesterol- and sphingolipid-rich membrane regions, populated by many molecules critical to receptor signaling 13
- 14. The Structure of Antibodies 14
- 15. • The identity between the binding sites of the secreted antibody and the membrane-bound B-cell receptor was first demonstrated by making reagents that bound to antibodies secreted by a particular clone of B cells and showing that those reagents also bound to the receptors on the cells that had secreted the antibodies. • Secreted antibodies and their membrane-bound receptor forms belong to the immunoglobulin family of proteins. • This large family of proteins, which includes both B- and T-cell receptors, adhesion molecules, some tyrosine kinases, and other immune receptors, is characterized by the presence of one or more 15
- 16. Antibodies Are Made Up of Multiple Immunoglobulin Domains • The immunoglobulin domain is generated when a polypeptide chain folds into an organized series of antiparallel beta-pleated strands. • In antibody molecules, most immunoglobulin domains contain approximately 110 amino acids, and each sheet contains 3-5 strands. • The pair of sheets within each domain are stabilized with respect to one another by an intrachain disulfide bond. 16
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- 18. • Neighboring domains are connected to one another by a stretch of polypeptide chain. • Within the strands, hydrophobic and hydrophilic amino acids alternate • The hydrophobic amino acids on one sheet are oriented toward the opposite sheet, and the two sheets within each domain are therefore stabilized by hydrophobic interactions between the two sheets as well as by the covalent disulfide bond. 18
- 19. • Immunoglobulin fold is adapted to provide a single scaffold onto which multiple different binding sites can be built, as the antigen-binding sites can simply be built into these loosely folded regions of the antigen- binding domains. • These properties explain why the immunoglobulin domain has been used in so many proteins with recognition or adhesive functions. • The essential domain structure provides a molecular backbone, while the loosely folded regions can be adapted to bind specifically to many adhesive or antigenic structures. 19
- 20. • Each of these proteins is classified as a member of the immunoglobulin superfamily, a term that is used to denote proteins derived from a common primordial gene encoding the basic domain structure. 20
- 21. Antibodies Structure • All antibodies share a common structure of 4 polypeptide chains consisting of two identical light (L) chains and two identical heavy (H) chains. • Each light chain is bound to its partner heavy chain by a disulfide bond between corresponding cysteine residues, as well as by noncovalent interactions between the VH and VL domains and the CH1 and CL domains. • These bonds enable the formation of a closely associated heterodimer (H-L). • Multiple disulfide bridges link the two heavy chains together about halfway down their length, and the C- terminal parts of the two heavy chains also participate in noncovalent bonding interactions between corresponding domains. 21
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- 23. • The antibody molecule forms a Y shape with two identical antigen-binding regions at the tips of the Y. • Each antigen-binding region is made up of amino acids derived from both the heavy- and the light chain amino-terminal domains. • The heavy and light chains both contribute two domains to each arm of the Y, with the non–antigen- binding domain of each chain serving to extend the antigen-binding arm. • The base of the Y consists of the C-terminal domains of the antibody heavy chain. 23
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- 25. • Overall structure of the antibody molecule consists of 3 relatively regions, joined by a more flexible hinge region. • The hinge region is particularly susceptible to proteolytic cleavage by the enzyme papain. • Papain cleavage resolves the antibody molecule into two identical fragments that retain the antigen-binding specificity of the original antibody and the remaining region of the molecule, which consists of the non antigen-binding portion. • This latter region, which is identical for all antibodies of a given class, crystallizes easily and was thus called the Fc region (fragment crystallizable). 25
- 26. • Each Fab region and Fc region of antibodies mediates its own particular functions during an antibody response to an antigen. • The Fab regions bind to the antigen, and the Fc region of the antigen-coupled antibody binds to Fc receptors on phagocytic or cytolytic cells, or to immune effector molecules. • In this way, antibodies serve as physiological bridges between an antigen present on a pathogen, and the cells or molecules that will ultimately destroy it. 26
- 27. Two Major Classes of Antibody Light Chains • Amino acid amino-terminal half (110 aa) of the light chain was extremely variable, whereas the sequence of the carboxyl-terminal half could be classified into one of two major sequence types. • N-terminal half of light chains is thus referred to as the variable, or VL, region of the light chain, and the less variable part of the sequence is termed the constant, or CL, region. • The two major light chain constant region sequences are kappa or lambda chains, lambda(λ) chain constant region sequences could be further subdivided into four 27
- 28. • In humans, the light chains are fairly evenly divided between the two light-chain classes; 60% of human light chains are κ whereas only 40% are λ . • In mice only 5% of mouse light chains are of the λ light- chain type. • All light chains have a molecular weight of 22 kDa. 28
- 29. • Within the variable regions of the light chain, there were regions of hypervariability. • Since these hypervariable regions could be shown to interact with the bound antigen, they were renamed the complementarity-determining regions (CDRs). 29
- 30. There are Five Major Classes of Antibody Heavy Chains • Each different heavy-chain constant region is referred to as an isotype, and the isotype of the heavy chains of a given antibody molecule determines its class. • Thus, antibodies with a heavy chain μ of the isotype are of the IgM class; those with a heavy chain δ are IgD; those withγ , IgG; those with ε, IgE; and those with α, IgA. • The length of the constant region of the heavy chains is either 330 amino acid residues (for gamma, delta, and alpha chains) or 440 amino acids (for mu and 30
- 31. • IgA, IgD, and IgG heavy chains weigh approximately 55 kDa, whereas IgM and IgE antibodies are approximately 20% heavier. • Minor differences in the amino acid sequences of groups of α and γ heavy chains led to further sub-classification of these heavy chains into sub-isotypes and their corresponding antibodies therefore into subclasses. • There are 2 sub-isotypes of the α heavy chain, 1 and 2, and thus two IgA subclasses, IgA1 and IgA2. • Similarly, there are four sub-isotypes of heavy chains, γ1, 2, 3, and 4, with the corresponding formation of the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. • The exact number, and precise positions of the disulfide bonds between the heavy chains of antibodies, vary among 31
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- 34. Each of the Domains of the Antibody Heavy and Light Chains Mediate Specific Functions • Antibodies protect the host against infection, by binding to pathogens and facilitating their elimination. • Antibodies of different heavy-chain classes are specialized to mediate particular protective functions, such as complement activation, pathogen agglutination, or phagocytosis, and each different domain of the antibody molecule plays its own part in these host defense mechanisms. 34
- 35. • CH1 and CL Domains • The strength (avidity) of receptor binding to antigen is greatly enhanced by receptor multivalency. • Antibodies have evolved to take advantage of this property by employing two antigen-binding sites, each of which can bind to individual determinants on multivalent antigens, such as are found on bacterial surfaces. • The CH1 and CL domains serve to extend the antigen- binding arms of the antibody molecule, facilitating interactions with multivalent antigens and maximizing the ability of the antibody to bind to more than one site on a multivalent antigen. 35
- 36. • The Hinge Regions • γ , δ, and α heavy chains contain an extended peptide sequence between the CH1 and CH2 domains that has no homology with the other domains. • Hinge region is rich in proline residues, rendering it particularly flexible, and as a consequence, the two antigen-binding arms of IgG, IgD, and IgA antibodies can assume a wide variety of angles with respect to one another, which facilitates efficient antigen binding. 36
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- 38. • In addition to these proline residues, the hinge region also contains a number of cysteines, which participate in heavy-chain dimerization. • The actual number of interchain disulfide bonds in the hinge region varies considerably among different heavy-chain classes and subclasses of antibodies as well as between species. • Lacking a hinge region, the heavy chains of IgE make their inter-heavy chain disulfide bonds between the CH1 and CH3 domains. • In IgM, disulfide bonds bridge the pairs of heavy chains at the level of CH3 and CH4. • Although M and E chains have no hinge regions, they do have an additional immunoglobulin domain that retains some hinge-like qualities. 38
- 39. Carbohydrate Chains • The two CH2 domains of α, δ, and γ chains and the two CH3 domains of μ and ε chains are separated from their partner heavy-chain domains by oligosaccharide side chains that prevent the two heavy chains from nestling close to one another. • As a result, the paired domains are significantly more accessible to the aqueous environment than other constant region domains. • This accessibility is thought to contribute to the ability of IgM and IgG antibodies to bind to complement components. 39
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- 41. The Carboxy-Terminal Domains The 5 classes of antibodies can be expressed as either membrane or secreted immunoglobulin. • Secreted antibodies have a hydrophilic amino acid sequence of various lengths at the carboxyl terminus of the final CH domain. • In membrane-bound immunoglobulin receptors, this hydrophilic region is replaced by three regions: 1. An extracellular, hydrophilic “spacer” sequence of approximately 26 amino acids 2. A hydrophobic transmembrane segment of about 25 amino acids 3. A very short, approximately 3 aa, cytoplasmic tail 41
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- 43. • Immature, pre-B cells express only membrane IgM. • Membrane IgD co-expression along with IgM is one of the markers of differentiation to a fully mature B cell that has yet to encounter antigen. • Following antigen stimulation, IgD is lost from the cell surface, and the constant region of the membrane and secreted immunoglobulin can switch to any one of the other isotypes. • The antibody class secreted by antigen-stimulated B cells is determined by cytokines released by T cells and APC in the vicinity of the activated B cell. • Antibodies of different heavy-chain classes have selective affinities for particular cell surface Fc receptors, as well as for components of the complement system. 43
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- 45. Signal Transduction in B Cells 45
- 46. • Prior to antigen recognition, mature B cells residing in the secondary lymphoid tissues, express membrane- bound forms of both IgM and IgD. • The cytoplasmic tail of the BCR heavy chain is extremely short so it is BCR associated with a heterodimer, Igα/Igβ, that is responsible for transducing the antigen signal into the interior of the cell. • BCR is structurally and functionally divided into 2 components: a recognition component and a signal transduction component. 46
- 47. T-Cell Receptors and Signaling 47
- 48. • When the T-cell receptor makes contact with its MHC-peptide antigen on the surface of APC, the 2 cell membranes are brought into close apposition with one another. 48
- 49. The T-Cell Receptor is a Heterodimer with Variable and Constant Regions • There are two types of T-cell receptors, both of which are heterodimers. The majority of recirculating T cells bear αβ heterodimers, which bind to ligands made up of an antigenic peptide presented in a molecular groove on the surface of a type I or type II MHC molecule. A second subset of T cells instead expresses a heterodimeric T-cell receptor composed of a different pair of protein chains, termed γ and δ . • T cells bearing γδ receptors have particular localization patterns and some T cells recognize different types of antigens from those bound by αβ T cells. 49
- 50. • Although some γδT cells recognize conventional MHC- presented peptide antigens, other γδ T cells bind lipid or glycolipid moieties presented by noncanonical MHC molecules. • Yet other γδ T-cell clones appear to recognize self- generated heat shock proteins or phosphoantigens derived from microbes • This ability of γδ T cells to break the rules of MHC restriction may account for the evolution of a slight difference in the angle between the antigen-binding and constant regions of the T-cell receptor, of the two types of receptor. 50
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- 52. • TCR proteins are members of the immunoglobulin superfamily of proteins and therefore the domain structures of TCR heterodimers are strikingly similar to those of the immunoglobulins. • α chain has a molecular weight of 40–50 kDa, and the β chain’s is 40–45 kDa. • Like the antibody light chains, the TCR chains have two immunoglobulin-like domains, each of which contains an intrachain disulfide bond spanning 60 to 75 amino acids. • The C domain of the TCR differs from most immunoglobulin domains in that it possesses only a single sheet, rather than a pair, and the remainder of the sequence is more variably folded. • The amino-terminal (variable) domain in both chains exhibits marked sequence variation, but the sequences of the remainder of each chain are conserved (constant). 52
- 53. • Each of the TCR variable domains has 3 hypervariable regions, which appear to be equivalent to the complementarity-determining regions in immunoglobulin light and heavy chains. • At the C-terminal end of the constant domain, each TCR chain contains a short connecting sequence, in which a cysteine residue forms a disulfide link with the other chain of the heterodimer. • C-terminal to this disulfide is a transmembrane region of 21 or 22 amino acids, which anchors each chain in the plasma membrane. 53
- 54. • The transmembrane domains of the TCR α and β chains are unusual in that they each contain positively charged amino acid residues that promote interaction with corresponding negatively charged residues on the chains of the signal transducing CD3 complex. • like BCRs, each TCR chain contains only a very short cytoplasmic tail at the carboxyl-terminal end. 54
- 55. The T-Cell Signal Transduction Complex Includes CD3 • TCR depends on a complex of proteins referred to collectively as CD3. • The CD3 complex is made up of three dimers: a (delta epsilon) pair, a (gamma epsilon) pair, and a third pair that is made up either of two CD3 (zeta) molecules or a (zeta, eta) heterodimer. • Each of the CD3 dimers contains negatively charged amino acids in its transmembrane domain that form ionic bonds with the positively charged residues on the intramembrane regions of the T-cell receptor. 55
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- 57. The T Cell Co-receptors CD4 and CD8 Also Bind the MHC • T-cell receptor is noncovalently associated with a • number of accessory molecules on the cell surface. • However, the only two such molecules that also recognize the MHC-peptide antigen are CD4 and CD8. 57
- 58. • CD4 is a 55 kDa monomeric membrane glycoprotein that contains four extracellular immunoglobulin-like domains (D1–D4), a hydrophobic transmembrane region, and a long cytoplasmic tail containing three serine residues that can be phosphorylated. • CD8 takes the form of a disulfide-linked αβ heterodimer orαα homodimer. • Both the αand β chains of CD8 are small glycoproteins of approximately 30 to 38 kDa. • Each chain consists of a single, extracellular, immunoglobulin-like domain, a stalk region, a hydrophobic transmembrane region, and a cytoplasmic tail containing 25 to 27 residues, several of which can be phosphorylated. 58
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- 60. • The extracellular domains of CD4 and CD8 bind to conserved regions of MHC class II and MHC class I molecules respectively. • Th e co-engagement of a single MHC molecule by both the TCR and its CD4 or CD8 coreceptor enhances the avidity of T-cell binding to its target. • This co-engagement also brings the cytoplasmic domains of the TCR/CD3 and the respective co- receptor into close proximity, and it helps to initiate the cascade of intracellular events that activate a T cell. 60
- 61. • Signaling through the antigen receptor, even when combined with that through CD4 or CD8, is insufficient to activate a T cell that has had no prior contact with antigen (a naïve T cell). • A naïve T cell needs to be simultaneously signaled through the TCR and its co-receptor, CD28, in order to be activated. • The TCR and CD28 molecules on a naïve T cell must co-engage the MHC-presented peptide and the CD28 ligand, CD80 (or CD86), respectively, on the antigen presenting cell for full activation to occur. 61