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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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