LECT 24 and 25: CELL SIGNAL TRANSDUCTION
Cells respond to molecular cues they receive: from neighboring cells or
extracellular matrices (paracrine factors), from the circulation (hormones
and metabolites), and from outside the body (sensory inputs).
Some messengers, e.g. nuclear hormones, directly permeate the cell
and interact with nuclear targets to effect responses.
Most messengers bind to cell surface receptors, which transduce signals
through intermediate proteins and messengers to ultimately effect changes
in the cytoplasm and in the nucleus. The signal is often amplified during
transduction, and feedback processes terminate or limit the response.
Cell surface signaling receptors largely fall into
several categories:
…7-TM G-protein-coupled receptors (GPCRs)
…Receptor protein kinases, including
receptor tyrosine kinases (RTKs)
…Others
Second Messengers Are Synthesized or Mobilized During Signaling
MANY SECOND MESSENGERS ACTIVATE SERINE/THREONINE PROTEIN KINASES
Each S/T-protein kinase phosphorylates exposed serine or threonine residues
within a consensus sequence. E.g.
PKA
------Arg-Arg-Xxx-Ser-Xxx-------O-
------Arg-Arg-Xxx-Ser-Xxx------ADP
HO-P=O
O-
ATP
7-TM GPCRs Are Numerous and Mediate Many Biological Functions
7-TM GPCRs Are Numerous and Mediate Many Biological Functions
GPCRs couple to heterotrimeric G proteins to mediate signaling
Activated b-adrenergic receptor activates Gas, which in turn
activates adenylate
cyclase (AC),
which synthesizes
2o messenger
cAMP, which
activates
protein kinase A
The Heterotrimeric G Protein Activation/Inactivation Cycle
Phospholipid Hydrolysis by PhospholipaseC Generates Two Second Messengers
There are several classes of PLC. One class is regulated by GPCRs
PLC-Derived Second Messengers Activate Protein Kinase C
ODORANT RECEPTOR STIMULATION OPENS cAMP-GATED CATION CHANNEL
Light-Activated Rhodopsin Turns Off a Dark Current Through
cGMP-gated Cation Channel
Rhodopsin is GPCR using light-activated retinal is ligand. Couples through
G protein transducin, which activates cGMP phosphodiesterase.
Insulin Receptor is Receptor Tyrosine Kinase Activated by
Insulin-Induced Receptor Autophosphorylation
Autophosphorylation is really receptor subunit transphosphorylation.
Autophosphorylation stimulates the receptor’s kinase activity AND
creates receptor recruitment sites for signaling target proteins.
IR Kinase Domain Phosphorylation Induces Conformational Change
When unphosphorylated, the “activation loop” of the kinase tends to fold
in such a way as to block the kinase’s ATP binding site.
Phosphorylation prevents this inhibition.
How does insulin induce IR phosphorylation? By two steps:
1. By changing the positioning of the two b subunits w.r.t. each other
2. Weak activity overcome by proximity of first target (other b subunit)
Tyrosine Phosphorylation Creates Target Recruitment & Activation Sites
IR phosphorylation in juxtamembrane region creates IRS-1 recruitment site.
Recruited IRS-1 is itself tyrosine phosphorylated by IR, creating a set
of recruitment sites.
PI3K is recruited to and activated by phosphorylated IRS-1.
PIP2 is phosphorylated by PI3K, which then activates protein kinase cascade.
Activated AKT mediates
many insulin responses
SH2 and PTB Domains of Target Protein Recognizes Phosphotyrosine in a
Sequence-Specific Context
The SH2 domain of PI3K recognizes the sequence -pY-x-x-M- on IRS-1.
The PTB domain of IRS-1 recognizes -L-Y-A-S-S-N-P-A-pY- on IR.
IRS-1 PTB bound to phospho-IR peptide
Epidermal Growth Factor Activates Its Receptor by Inducing
Dimerization and Autophosphorylation
Monomeric EGFR is dimerized by EGF, by stabilizing and intrinsic
receptor dimerization site.
Dimerization brings kinase domains close together, enabling
auto(trans)phosphorylation, creating substrate recruitment sites.
Ligand-mediated
receptor clustering
is a common
theme in signal activation
Epidermal Growth Factor Receptor Signals to Activate RAS Protein
Oncogenic RAS mutations stabilize the
GTP-bound state in absence of growth
factor, causing unregulated signaling
Notch Receptor Signaling Controls Local Cell Fate Decisions
-- Notch (a receptor) and its ligands Delta and Serrate are conserved in all
vertebrates as well as complex invertebrates (flies, worms, etc).
-- Notch controls cell fate decisions at many times and places in development.
-- The most classic type of decision process mediated by Notch is
Lateral Inhibition: a group of equipotent cells selects some to assume a
specific fate, while others of the group are inhibited. Inhibition requires Notch.
Normal
Precursor
NotchDevelopment
Ablation
Mutant
Uncommited
Precursors
Differentiated
Cells
-- Ligand for Notch produced in cell assuming “black” fate, acting to inhibit
neighbors from assuming same fate.
-- Notch signaling thereby induces the “red” differentiated state
Canonical Notch Signaling Pathway
Transmembrane Notch binds
a membrane-bound ligand
(Delta or Serrate) on
neighboring cell.
Induces Notch proteolysis,
freeing cytoplasmic NICD.
NICD goes to nucleus, acting
as cofactor for activation
transcription.
Signaling is terminated by
NICD phosphorylation,
ubiquitination, and
proteasomal degradation.
The Logic of Notch-Mediated Lateral Inhibition
Equipotent precursors may have
random small differences in
Notch and Delta expression levels.
Notch signaling mediates a pathway
through E(spl) and AS-C that
enhances Notch expression and
decreases Delta expression
Loss of Notch signaling decreases
Notch expression and enhances
Delta expression.
The AS-C transcription factor also
controls adoption of a particular
differentiated state, so that
Notch signaling blocks this
differentiated state.
Fringe-Mediated Notch Glycosylation Alters Ligand Binding to Notch
Development of adult appendages (e.g. wings) in insects occurs in imaginal
discs that are patterned during larval stages. A key step in patterning is
activation of Notch in a 2-cell-width stripe along dorsal/ventral midline of disc.
Fringe is a cell-bound Notch glycosidase
that shifts Notch’s ligand preference from Serrate to Delta
At early stage of disc patterning,
Notch is expressed everywhere,
Delta is not expressed, and
Serrate and Fringe are only
in dorsal half of disc.
Serrate can only activate
unmodified Notch at
ventral margin.
Serrate also induces Delta
expression in ventral margin.
Delta can then activate
modified Notch at
dorsal margin.