B- and T-

Cell Lecture 4A

Receptors
Recall
• A cellular signal is any event that instructs a cell to
change its metabolic or proliferative state
• Signals are usually generated by the binding of a
ligand to a complementary cell-bound receptor
• A cell can become more or less susceptible to
actions of a ligand by increasing or decreasing
expression of the receptor for that ligand
Recall
• The ligand may be a soluble molecule or a
peptide, carbohydrate, or lipid presented
on the cell surface
• The ligand may travel long distances from
its entry point in either the bloodstream
or lymphatics before it reaches a cell
bearing the relevant receptor
• Ligand-receptor binding is noncovalent,
although it may be of quite high affinity
Recall
• Ligand-receptor binding induces molecular
change in the receptor
• Conformational
• Dimerization/clustering
• Location in the membrane
• Covalent modification
• Receptor alterations induce cascades of
intracellular events
• Activation of enzymes
• Changes in intracellular locations of
molecules
Recall
• Cell-signaling end results often induce a
change in the transcriptional program of
the target cell
• Sometimes multiple signals through
multiple receptors are required to effect
particular outcomes
• Integration of all signals received by a cell
occurs at the molecular level inside the
recipient cell
 Receptor-ligand interactions
• Receptor-ligand binding occurs
via multiple noncovalent bonds
• Each individual bond may be
weak
• Many such bonds occur between
receptors and ligands, providing
great cumulative bond strength
Receptor-ligand
interactions
• Antigen-immune system receptor interactions are
enhanced by co-receptor binding
• These are separate receptor-ligand interactions that
may take place near the original interaction
• Often times, a single type of interaction may be
insufficient to lead to an activation event
• A co-receptor interaction may provide a second
signaling interaction to further signal the cell to
proceed with activation
Receptor-ligand interactions
• Receptor-antigen interactions are usually multivalent
• Multivalency increases avidity of the interactions
• Individual interactions have an affinity―a strength
of that individual pairing
• Avidity is the combined strength of multiple
interactions
• As such, an interaction may have weak affinity, but
high overall avidity
 Receptor-ligand interactions

• Receptor and ligand expression can vary during the course

of an immune response
• An example: white blood cells (blue) treated with an activating
mitogen show upregulation of the receptor for cytokine IL-2
(yellow-orange)
 Receptor-ligand interactions
• Local concentrations of cytokines and other ligands may be
extremely high
• A cell may direct its secretion machinery toward a recipient for
maximum effect
• An example: blue dendritic cells secreting cytokine IL-12 (pink) to
T cells (green); note the localization of the cytokine-filled, vesicle-
rich area in the dendritic cell
 • Binding of antigen to receptor
induces:
• An internal signaling cascade,
Common which:
• Leads to cellular alterations in:
stages • Motility
• Adhesive properties
used in • Transcriptional
programming
many • These cascades are behind the various
signaling cellular changes that take place during
an immune response against an
pathways antigen
• Often, the same players/proteins are
used in different cell types―triggering
receptors may be different
 Common stages used in many
signaling pathways
• Antigen-mediated receptor clustering initiates signaling in
B and T cells
• Receptor dimerization (shown in figure) is often a result
• Multimerization
can also occur
• Clustered receptors
are localized in
lipid rafts
 Common stages used in many
signaling pathways
• Antigen-mediated receptor clustering initiates signaling in
B and T cells
• Receptor dimerization is often a result
• Multimerization can also occur
• Clustered receptors are localized in lipid rafts
 Common stages used in many
signaling pathways
• Some receptors require receptor-associated molecules to
signal cell activation
• B- and T-cell receptors have short cytoplasmic portions
• Other molecules associate with them for signal transduction
• B cells use Igα and Igβ molecules (among others)
• T cells use CD3 complexes and CD28 molecules
 Common stages used in many
signaling pathways
• Tyrosine phosphorylation is an early step in many signaling
pathways
• CD3 (T cells) and Igα/β (B cells) are phosphorylated on ITAMs
• Immunoreceptor tyrosine activation motifs
• P-tyrosine serve as docking points for adapter molecules
• Src-family kinases
phosphorylate tyrosines
• These kinases become
activated themselves when
phosphorylated
Common stages used in many
signaling pathways
• Adapter proteins help to gather
members of signaling pathways
• Phosphorylation of serine/threonine
residues is also a common step in
signaling pathways with different
possible outcomes
• May activate an enzyme
• Induce interaction with other proteins
• Alter cellular location
• Protect protein from destruction
• Target the protein for destruction
Common stages used in many
signaling pathways
• Adapter proteins help to gather
members of signaling pathways
• Phosphorylation of membrane
phospholipids recruits pleckstrin
homology (PH) domain-containing
proteins to the membrane
• PIP2 may be phosphorylated by phosphatidyl
inositol-3-kinase (PI3 kinase)
• This creates PIP3 (diagram), which can bind
proteins with PH domains
 Common stages used in many
signaling pathways
• Adapter proteins help to gather members of signaling
pathways
• Signal-inducing PIP2 breakdown by phospholipase C (PLC) causes
an increase in cytoplasmic calcium ion concentration
 Common stages used in many
signaling pathways
• Adapter proteins help to
gather members of
signaling pathways
• Calcium ions can then bind
several cellular proteins,
changing their
conformation and altering
activity
• Calmodulin (CaM) is an
important member of this
group
 Frequently encountered signaling
pathways
• The PLC pathway induces calcium
release and Protein Kinase C (PKC)
activation
• Diagram illustrates the early
(membrane-localized) and late
(cytoplasmic/nuclear) events
• Overall result of released calcium ions is
activation of the nuclear factor of activated
T cells (NFAT) transcription factor
• Diacylglycerol (DAG) left behind also
facilitates activation of PKC for activation of
a different transcription factor
 Frequently encountered signaling
pathways
• The Ras/MAP kinase cascade activates
transcription through AP-1
• Ras is a G protein
• It becomes active when it swaps a GTP molecule
in for its GDP molecule
• Guanine-nucleotide exchange factors (GEFs)
activate Ras by inducing this swap
• GTPase activating proteins (GAPs) inhibit Ras by
stimulating its ability to break down GTP into
GDP
• Ras (once active) participates in
downstream signaling events
Frequently encountered signaling
pathways
• The Ras/MAP kinase cascade activates
transcription through AP-1
• DAG recruits the adapter protein Ras-GRP
• Ras-GRP recruits SOS (a GEF) which helps to
activate Ras
• Ras then activates a series of serine/threonine
kinases known as the mitogen activated protein
kinase (MAPK) cascade
• The end of this cascade results in the
phosphorylation and nuclear transport of
extracellular signal-related kinase (ERK)
Frequently encountered signaling
pathways
• The Ras/MAP kinase cascade activates
transcription through AP-1
• Nuclear events
• pERK phosphorylates and activates the Elk-1
transcription factor
• This leads to production of the Fos protein
• Fos becomes phosphorylated by ERK and binds
with phosphorylated Jun protein
• This pJun/pFos combination is known as master
transcription factor AP-1
• AP-1 can facilitate transcription of the IL-2 gene,
and important cytokine for immune responses
 Frequently encountered signaling
pathways
• PKC activates NF-κB
• Nuclear factor kappa B
• Heterodimeric family of transcription factors
• Held inactive in the cytoplasm, bound with
inhibitor of NF-κB (IκB) protein
• Cell activation phosphorylates the inhibitor
proteins using an IκB kinase (IKK) complex
• DAG activates PKC, activating IKK complexes
• Phosphorylation/ubiquitination of IκB
targets it for destruction
• now-active NF-κB translocates to the
nucleus to enhance transcription
 The structure of antibodies

• The antigen receptor on B cells is a membrane-bound form

of immunoglobulin secreted upon stimulation
• Multiple immunoglobulin domains
 The structure of antibodies
• Immunoglobulin = antibody
• Two heavy chains (variable and constant regions)
• Two light chains (variable and constant regions)
• Held together by intra-/interchain disulfide covalent bonds
 The structure of antibodies
• Secreted antibodies are
grouped into five major
classes
• Differentiated by amino
acid sequence of heavy
chain
• Each class performs
different functions
during immune
responses
 • Each of the domains of Ab
heavy/light chains mediate
specific functions
The • CH1 and CL domains
• Extend the “arms” of the antibody
structure away from the hinge region
• VH and VL domains
of • Two particular sites formed at the
antibodies “top of the Y” of the antibody
• Produce the ability of the Ab
molecule to bind to its specific
antigen
• Each Ab can bind two antigen
molecules
 The structure of antibodies

• Each of the domains of Ab heavy/light chains mediate

specific functions
• Hinge regions
• Found in between CH1 and CH2 regions
• Rich in proline residues, making it flexible
• Allows antigen-binding “arms” of Ab to flex inward and outward
• Also rich in cysteines, facilitating heavy-chain dimerization through
interchain disulfide bond formation
The structure of antibodies
• Each of the domains of Ab
heavy/light chains mediate specific
functions
• Carbohydrate chains
• Antibodies are generally quite glycosylated
• Some heavy-chain domains are separated
by oligosaccharide side chains that help
“spread” the domains apart
• Other antibodies have carbohydrates
attached to light-chain domains
 The structure of antibodies
• Each of the domains of Ab heavy/light chains mediate
specific functions
• Carboxy-terminal domains
• Membrane-bound Ab has three “extra” regions
• An extracellular hydrophilic 26 amino acid “spacer”
• A 25 amino acid hydrophobic
transmembrane segment
• A very short three amino acid
cytoplasmic tail
• Secreted Ab is formed by
alternative RNA splicing
mechanisms that remove/replace
these regions
 Signal transduction in B cells
• Antigen signaling in B cells
proceeds according to
signaling strategies shared
among many cell types
• Differences exist near the
plasma membrane
• Past these early events, steps
for each pathway are largely
similar
 T-cell receptors and signaling

• T-cell receptors (TCRs) are not immunoglobulins

• But they have Ig domains
• They belong to the Ig superfamily of proteins
• They possess variable (V) domains and constant (C) domains, just
as in Ab molecules
 T-cell receptors and signaling
• TCRs are heterodimers
• Most possess both an α (alpha) and a β (beta) chain
• Recognize a short peptide carried in the groove of a major
histocompatibility complex (MHC) protein on the surface of an
antigen-presenting cell (APC)
• TCRs work as a part of a complex that includes CD3
T-cell • TCRs are heterodimers
receptors • A small subset of T cells carry a γδ
(gamma delta) receptor instead
and • These cells recognize antigens in a
different manner than αβ T cells
signaling
T-cell receptors
and signaling
• The T-cell co-receptors CD4 and
CD8 also bind the MHC to aid
signal transduction
• CD4 is a 55 kDa monomer
• Four extracellular Ig
domains
• Binds to regions on
MHC Class II
• CD8 is usually a
heterodimer
• Disulfide-linked α and β
chains
• Each chain is 30-38 kDa
• Binds to regions on
MHC Class I
T-cell receptors and signaling
• Several different T-cell accessory molecules assist in
T-cell signal transduction
T-cell receptors
and signaling
• Lck (lymphocyte-specific protein
tyrosine kinase) is the first
tyrosine kinase activated in T-
cell signaling
• Differences from B-cell
pathways exist near the
plasma membrane
• Past these early events,
steps for each pathway are
largely similar
• Antigen signaling in T cells
shares many characteristic
strategies with B-cell
signaling
Summary
• Signal transduction is a complex subject
• The trick is to look for similarities and differences
• Both B and T lymphocytes use similar strategies once
the processes are underway
• The differences lie at the level of the antigen receptors
and the early membrane-linked events
• By understanding antigen receptor and early
membrane event differences, we can better
understand how signaling (and activation) works in
each lymphocyte cell type