Chapter 16, see p330 in text book
Cell-to-Cell Signaling:
Hormones and Receptors
signaling molecules
receptors
signal transduction
Overview of Extracellular Signaling
(1) synthesis and (2) release of the signaling molecule;
(3) transport of the signal to the target cell;
(4) detection of the signal by a specific receptor protein;
(5) a change in cellular metabolism, function, or
development triggered by the receptor-signal complex;
(6) removal of the signal, which often terminates the cellular
response.
1.1 Signal molecules:
Signaling Molecules Operate over Various
Distances in Animals
1) endocrine, 2) paracrine, or autocrine
3) Synaptic signal
In endocrine signaling, signaling molecules,
called hormones, act on target cells distant
from their site of synthesis by cells of
endocrine organs.
Receptor Proteins Exhibit Ligand-Binding and
Effector Specificity
Hormones Can Be Classified Based on Their
Solubility and Receptor Location
Most hormones: (1) small lipophilic molecules that
diffuse across the plasma membrane and interact with
intracellular receptors; and (2) hydrophilic or (3)
lipophilic molecules
Lipophilic Hormones with Intracellular
Receptors
steroids (cortisol, progesterone, estradiol, and
testosterone), thyroxine, and retinoic acid
Water-Soluble Hormones with Cell-Surface
Receptors
(1)peptide hormones, such as insulin, growth
factors, and glucagon, and (2) small charged
molecules, such as epinephrine and histamine
Lipophilic
Receptors
Hormones
with
Cell-Surface
Cell-Surface Receptors Belong to Four Major
Classes
G protein coupled receptors
Ion-channel receptors
Tyrosine kinase linked receptors
Receptors with intrinsic enzymatic activity
Effects of Many Hormones Are Mediated by
Second Messengers
second messengers,intracellular signaling molecules, cAMP;
cGMP; 1,2-diacylglycerol (DAG); IP3; various inositol
phospholipids (phosphoinositides); and Ca2+.
Other Conserved Proteins Function in Signal
Transduction
GTPase Switch Proteins: there are two classes of GTPase
switch proteins, and monomeric Ras and Ras-like proteins.
Protein Kinases
Adapter Proteins
Common Signaling Pathways Are Initiated by
Different Receptors in a Class
The Synthesis, Release, and Degradation of
Hormones Are Regulated
Peptide Hormones and Catecholamines
Steroid Hormones, Thyroxine, and Retinoic Acid
Feedback Control of Hormone Levels
SUMMARY
signaling molecules, membrane-anchored and secreted
proteins, lipophilic and hydrophilic molecules, and gases.
Binding of extracellular signaling molecules to cell-surface
receptors trigger intracellular pathways that modulate
cellular metabolism, function, or development.
The second messengers, such as Ca2+, cAMP, and IP3 ;
Conserved proteins in signal-transduction pathways
include GTPase switch proteins, protein kinases, and
adapter proteins.
Extracellular signals are often integrated into complex
regulatory networks in which the synthesis, release, and
degradation of hormones are precisely regulated.
Identification and Purification of CellSurface Receptors
Binding of a hormone to a receptor involves
of weak interactions ionic and van der Waals
bonds and hydrophobic interactions.
Hormone Receptors Are Detected by Binding
Assays
The KD, the hormone concentration at which the receptor
is half-saturated, also can be calculated from the specific
binding curve. KD Values for Cell-Surface Hormone
Receptors Approximate the Concentrations of Circulating
Hormones
Affinity Techniques Permit Purification of
Receptor Proteins
Many Receptors Can Be Cloned without Prior
Purification
SUMMARY
Receptors bind to ligands. This specificity is
determined by interactions between ligand
determinants and specific amino acids in the receptor.
Receptors can be purified directly using ligands as
affinity reagents.
In some cases, the genes encoding receptors for
specific ligands can be isolated from cDNA libraries
transfected into cultured cells. Cells expressing the
receptor are detected using labeled ligand as a probe.
G Protein Coupled Receptors and Their Effectors
cell-surface receptors --- trimeric signal--transducing G protein---effector enzyme --- an
intracellular second messenger
All G protein coupled receptors (GPCRs) contain
seven membrane-spanning regions with N-terminal
segment on the exoplasmic face and C-terminal
segment on the cytosolic face of the plasma
membrane.
Hormone—receptor—G protein—enzyme—the
second message—kinase—enzyme or functional
protein—biological effect
Binding of Epinephrine to Adrenergic
Receptors Induces Tissue-Specific Responses
Stimulation of b-Adrenergic Receptors Leads
to a Rise in cAMP
Critical Features of Catecholamines and Their
Receptors Have Been Identified
Trimeric Gs Protein Links b-Adrenergic
Receptors and Adenylyl Cyclase
b-adrenergic receptors, which are coupled to Gs,
or stimulatory G protein
b-adrenergic receptors is an elevation in the
intracellular level of cAMP.
Cycling of Gs between Active and Inactive Forms
The G proteins and other GPCRs contain three subunits
designated a, b, and g. GTPase switch proteins alternate
between an "on" state with bound GTP and an "off" state.
Binding of a hormone or agonist to the receptor changes its
conformation, causing it to bind to the trimeric Gs protein
in such a way that GDP is displaced from Gsa and GTP is
bound. The Gsa ·GTP complex, which dissociates from the
Gbg complex, then binds to and activates adenylyl cyclase.
Amplification of Hormone Signal
Termination of Cellular Response
Some Bacterial Toxins Irreversibly Modify G
Proteins
Adenylyl Cyclase Is Stimulated and Inhibited
by Different Receptor-Ligand Complexes
GTP-Induced Changes in Gsa Favor Its Dissociation
from G b g and Association with Adenylyl Cyclase
Gia and Gsa Interact with Different Regions of
Adenylyl Cyclase
Degradation of cAMP Also Is Regulated
SUMMARY
Many cell-surface receptors, seven transmembrane domains
- trimeric G proteins.
All G proteins contain three subunits: a, b, and g.
The intrinsic GTPase activity of G a inactivates G a · GTP by
catalyzing GTP hydrolysis: Pi is released and the resulting G
a · GDP then dissociates from its effector and reassociates
with G b g .
Adenylyl cyclase, which catalyzes the formation of cAMP
from ATP, is the best-characterized effector regulated by
trimeric G proteins. All adenylyl cyclase isoforms are
stimulated by Gsa , but only specific isoforms are inhibited
by Gia and G b g . Gsa , Gia , and G b g interact with different
regions of the catalytic domain of adenylyl cyclase.
Receptor Tyrosine Kinases and Ras
receptor tyrosine kinases (RTKs) , The ligands for RTKs
are soluble or membrane-bound peptide/protein hormones
including nerve growth factor (NGF), platelet-derived
growth factor (PDGF), fibroblast growth factor (FGF),
epidermal growth factor (EGF), and insulin.
All RTKs comprise an extracellular domain containing a
ligand-binding site, a single hydrophobic transmembrane
a helix, and a cytosolic domain that includes a region
with protein-tyrosine kinase activity. Binding of ligand
causes most RTKs to dimerize; the protein kinase of
each receptor monomer then phosphorylates a distinct
set of tyrosine residues in the cytosolic domain of its
dimer partner, a process termed autophosphorylation.
The phosphotyrosine residues in activated RTKs interact
with adapter proteins containing SH2 or PTB domains.
Ras and G a Subunits Belong to the GTPase
Superfamily of Intracellular Switch Proteins
Activation of both Ras and G a is triggered by hormone
binding to an appropriate cell-surface receptor. Ras
activation is accelerated by a protein called guanine
nucleotide exchange factor (GEF), which binds to the
Ras · GDP complex, causing dissociation of the bound
GDP.
Hydrolysis of the bound GTP deactivates both Ras and G a . The
average lifetime of a GTP bound to Ras is about 1 minute, which is
much longer than the lifetime of G a · GTP. The reason for this
difference is that the deactivation of Ras, unlike the deactivation of G
a , requires the assistance of another protein: a GTPase-activating
protein (GAP), which binds to Ras · GTP and accelerates its intrinsic
GTPase activity by a hundredfold.
An Adapter Protein and GEF Link Most
Activated RTKs to Ras
SH2 Domain in GRB2 Adapter Protein Binds
to a Specific Phosphotyrosine in an Activated
RTK
Sos, a Guanine Nucleotide Exchange Factor,
Binds to the SH3 Domains in GRB2
SUMMARY
Receptor tyrosine kinases (RTKs), bind to peptide hormones, may
exist as dimers or dimerize during binding to ligands.
Ligand binding leads to activation of the kinase activity of the
receptor and autophosphorylation of tyrosine residues.
Ras is an intracellular GTPase switch protein that acts downstream
from most RTKs. Like Gsa , Ras cycles between an inactive GDPbound form and active GTP-bound form.
Unlike GPCRs, which interact directly with an associated G protein,
RTKs are linked indirectly to Ras via two proteins, GRB2 and Sos.
The SH2 domain in GRB2, an adapter protein, binds to specific
phosphotyrosines in activated RTKs.
Normally, Ras activation and the subsequent cellular response is
induced by ligand binding to an RTK. However, in cells that
contain a constitutively active Ras, the cellular response occurs in
the absence of ligand binding.
MAP Kinase Pathways
Activated Ras then induces a kinase cascade that culminates in
activation of MAP kinase. This serine/threonine kinase, which can
translocate into the nucleus, phosphorylates many different proteins
including transcription factors that regulate expression of important
cell-cycle and differentiation-specific proteins.
Signals Pass from Activated Ras to a Cascade of Protein Kinases
Ksr May Function as a Scaffold for the MAP Kinase Cascade
Linked to Ras
Phosphorylation of a Tyrosine and a Threonine Activates
MAP Kinase
Various Types of Receptors Transmit Signals to MAP Kinase
Multiple MAP Kinase Pathways Are Found in Eukaryotic
Cells
Specificity of MAP Kinase Pathways Depends on
Several Mechanisms
SUMMARY
Activated Ras promotes formation of signaling complexes at the
membrane containing three sequentially acting protein kinases and a
scaffold protein Ksr. Raf is recruited to the membrane by binding to
Ras · GTP and then activated. It then phosphorylates MEK, a dual
specificity kinase that phosphorylates MAP kinase. Phosphorylated
MAP kinase dimerizes and translocates to the nucleus where it
regulates gene expression.
RTKs, GPCRs, and other receptor classes can activate MAP kinase
pathways.
In MAP kinase pathways containing common components, the
activity of shared components is restricted to only a subset of MAP
kinases by their assembly into large pathway-specific signaling
complexes.
Some MAP kinases have kinase-independent functions that can
restrict signals to only a subset of MAP kinases.
The pathway of signal transduction,
Second Messengers
cAMP and Other Second Messengers Activate Specific
Protein Kinases
1) cAMP-dependent protein kinases (cAPKs)
cAPKs Activated by Epinephrine Stimulation Regulate
Glycogen Metabolism
Kinase Cascades Permit Multienzyme Regulation and
Amplify Hormone Signals
effector enzyme adenylyl cyclase, which
synthesizes the second messenger cAMP.
Phosphodiesterase, PDE
The cAMP-dependent protein kinases are tetramers, consisting of
two regulatory (R) subunits and two catalytic (C) subunits.
Binding of cAMP to the R subunits causes dissociation of the two
C subunits, which then can phosphorylate specific acceptor
proteins.
Cellular Responses to cAMP Vary among Different
Cell Types
The effects of cAMP on the synthesis and degradation of glycogen
are confined mainly to liver and muscle cells, which store
glycogen. However, cAMP also mediates the intracellular
responses of many other cells to a variety of hormones that
stimulate Gs protein coupled receptors. The effects of cAMP on a
given cell type depend, in part, on the specificity of the particular
cAPK and on the cAPK substrates that it expresses.
Anchoring Proteins Localize Effects of cAMP to
Specific Subcellular Regions
Modification of a Common Phospholipid Precursor
Generates Several Second Messengers
The inositol group in phospholipid, which extends into
the cytosol adjacent to the membrane, can be [reversibly]
phosphorylated at various positions by the combined
actions of various kinases and phosphatases. The levels of
PIs in cells are dynamically regulated by extracellular
signals. In response to some signals (e.g., PDGF), there is
an acute rise in PIs phosphorylated at this position
through the activation of PI-3 kinase.
Phosphoinositides can be cleaved by the
membraneassociated enzyme phospholipase C (PLC) to
generate yet other second messengers. These cleavage
reactions produce 1,2-diacylglycerol (DAG), a lipophilic
molecule that remains linked to the membrane, and free
phosphorylated inositols, which can diffuse into the
cytosol. The main pathway generates DAG and inositol
1,4,5-trisphos-phate (IP3). Signaling pathways involving
any of these second messengers sometimes are referred to
as inositol-lipid pathways.
Hormone-Induced Release of Ca2+ from the
ER Is Mediated by IP3
Opening of Ryanodine Receptors Releases
Ca2+ Stores in Muscle and Nerve Cells
Ca2+-Calmodulin Complex Mediates Many Cellular
Responses
A small cytosolic protein called calmodulin, which is
ubiquitous in eukaryotic cells, mediates many cellular
effects of Ca2+ ions. Ca2+-calmodulin complex is cAMP
phosphodiesterase; this enzyme degrades cAMP
regulation and activates several protein kinases that, in
turn, phosphorylate transcription factors, thereby
modifying their activity and regulating gene expression.
DAG Activates Protein Kinase C, Which Regulates
Many Other Proteins
Synthesis of cGMP Is Induced by Both Peptide
Hormones and Nitric Oxide
SUMMARY
Second messengers activate certain protein kinases.
Phosphorylation of a specific region of the catalytic
domain called the phosphorylation lip further activates
these protein kinases.
cAMP-dependent protein kinases (cAPKs) mediate the
diverse effects of cAMP in different cells. The effect of
cAMP in a cell depends largely on the particular cAPK
and the protein substrates that it contains.
In liver and muscle cells, hormone-induced activation of
cAPK sets into motion a kinase cascade that both inhibits
glycogen synthesis and stimulates glycogen breakdown.
Kinase cascades triggered by cAPK and other second
messenger controlled protein kinases can regulate multiple
target proteins. Localization of cAPK to specific regions of
the cell by anchoring proteins restricts the effects of cAMP
to particular subcellular locations.
Signaling through GPCRs and RTKs stimulates PI-3
kinase to generate specific phosphoinositides. Activation
of both GPCRs and RTKs activate phospholipase C, which
hydrolyzes PIP2 to the second messengers IP3, which
diffuses into the cytosol, and DAG, which remains
membrane bound.
Hormone stimulation of the inositol-lipid pathway leads to the IP3mediated release of Ca2+ ions and activation of protein kinase C by
DAG. Ca2+ forms a complex with a multivalent Ca2+- binding protein
called calmodulin. The Ca2+-calmodulin complex regulates the
activity of many different proteins, including protein kinases that in
turn regulate the activity of various transcription factors.
Protein kinase C is coordinately regulated by Ca2+, which recruits it
to the membrane, and DAG, which activates it.
cGMP is produced by cell-surface receptors with guanylate cyclase
activity, which are activated by peptide hormones, and by soluble
guanylate cyclase, which is activated by binding of nitric oxide.
Interaction and Regulation of Signaling
Pathways
The Same RTK Can Be Linked to Different Signaling
Pathways
Multiple G Proteins Transduce Signals to Different Effector
Proteins
G b g Acts Directly on Some Effectors in Mammalian Cells
Glycogenolysis Is Promoted by Multiple Second Messengers
Molecular Mechanisms of Signal Transduction
cAMP as the second messenger-which mediates the
cellular response to epinephrine; other fundamental
hormone mechanisms, involving different second
messengers (cGMP, DAG, IP3, Ca2+), a protein-tyrosine
kinase activity, and ligand- and voltage-activated ion
channels; The phosphorylation and dephosphorylation of
specific proteins are shown to be central to these
mechanisms; steroid hormones function through the
regulation of gene activity;
Receptors for Epinephrine Trigger Cyclic AMP
Production
Cyclic AMP Acts as a Second Messenger for a Number
of Regulatory Molecules
Cyclic GMP Also Acts as a Second Messenger
The Insulin Receptor Is a Tyrosine-Specific Protein
Kinase
Two Second Messengers Are Derived from
Phosphatidylinositols
Calcium Is a Second Messenger in Many Signal
Transductions
Ion Channels Are Gated by Ligands and by
Membrane Potential
Toxins, Oncogenes, and Tumor Promoters
Interfere with Signal Transductions
Steroid and Thyroid Hormones Act in the
Nucleus to Change Gene Expression
Insulin Stimulation Activates MAP Kinase and Protein
Kinase B
Ras-Dependent Pathway
Ras-Independent Pathway
Insulin and Glucagon Work Together to Maintain a
Stable Blood Glucose Level
Receptors for Many Peptide Hormones Are DownRegulated by Endocytosis
Phosphorylation of Cell-Surface Receptors Modulates
Their Activity
Arrestins Have Two Roles in Regulating G Protein
Coupled Receptors
SUMMARY
Many RTKs and GPCRs activate multiple signaling pathways, and
different second messengers sometimes mediate the same cellular
response.
Some activated RTKs are coupled to the Ras-MAP kinase pathway or
inositol-lipid pathway in a tissuespecific manner.
Eukaryotes possess multiple G a , G b , and G g subunits. Different G a
subunits activate various effector proteins, leading to production of
specific second messengers .
The activity of some effector proteins, including certain adenylyl
cyclase isoforms, is regulated by G b g .
Glycogen breakdown and synthesis is regulated by multiple second
messengers induced by neural or hormonal stimulation.
The insulin receptor, a dimeric RTK, can act through a Ras-dependent
pathway, leading to activation of MAP kinase, or through a Rasindependent pathway involving phosphoinositides, leading to activation
of protein kinase B.
Insulin stimulation of muscle cells and adipocytes leads to activation of
protein kinase B, which promotes glucose uptake and glycogen
synthesis, resulting in a decrease in blood glucose.
Binding of glucagon to its GPCR promotes glycogenolysis and an
increase in blood glucose via the cAMP-triggered kinase cascade.
Ligand binding frequently induces phosphorylation of the
cytosolic domain of a cell-surface receptor, thereby modulating
its activity.
At high ligand concentration, some cell-surface receptors are
internalized by endocytosis, reducing the number of receptors on
the surface and making cells less sensitive to ligand.
Many internalized RTKs are degraded in lysosomes. In this case,
resensitization depends on synthesis of additional receptor
molecules.
Internalization of phosphorylated (inactive) GPCRs leads to
receptor dephosphorylation, b-arrestin dissociation, and recycling
of active receptors to the cell surface.
From Plasma Membrane to Nucleus
In one cell surface → nucleus signaling pathway, binding of
interferon g to its cell-surface receptor induces membrane
recruitment and activation of a cytosolic protein-tyrosine
kinase called JAK. The activated JAK then phosphorylates
Stat1, a member of transcription factors. Cytosolic Smad
transcription factors are activated by receptor serine/threonine
kinases that bind growth factors in the TGFb superfamily.
The cAMP and MAP kinase pathways, activated cytosolic
kinases translocate to the nucleus where they directly modify
transcription factors.
CREB Links cAMP Signals to Transcription
In mammalian cells, an elevation in the cytosolic cAMP
level stimulates the expression of many genes.
cAMP-response element (CRE) binds the phosphorylated
form of a transcription factor called CRE-binding (CREB)
protein
MAP Kinase Regulates the Activity of Many
Transcription Factors
Phosphorylation-Dependent
Regulates NF-kB
Protein
Degradation
NF-kB is a heterodimer of two related proteins of 65 kD
and 50 kD. The proteins share a region of homology at
their N-termini that is required for DNA binding and
dimerization. In response to an extracellular signal, NFkB translocates to the nucleus, where it binds to specific
sites in DNA and regulates transcription.
NF-kB is sequestered in an inactive state in the cytoplasm
by direct binding to an inhibitor called I-kB. In response to
signal, I-kB is phosphorylated at two N-terminal serine
residues by an I-kB kinase complex. Phosphorylated I-kB is
targeted for ubiquitination and degraded in the proteosome.
Phosphorylation-dependent degradation of the cyclin-kinase
dependent inhibitor, Sic 1, plays a central role in regulation.
It seems that phosphorylation-dependent protein degradation
may emerge as a common regulatory mechanism in many
different cellular processes.
SUMMARY
Protein phosphorylation plays a key role in regulating
transcriptional activity in response to specific extracellular signals.
The catalytic subunit of cAMP-dependent protein kinase
translocates to the nucleus, where it phosphor-ylates CREB protein,
which then interacts with the coactivator CBP/P300. The resulting
trimeric complex binds to and activates transcription of target genes
containing the CRE sequence.
CBP/P300 also physically interacts with transcription factors whose
activity is modulated by other signaling pathways.
MAP kinase, activated via the RTK-Ras pathway, translocates to the
nucleus, where it phosphorylates various transcriptional activators
and repressors. MAP kinase phosphorylation promotes the activity
of some transcription factors and inhibits the activity of others.
NF-kB is localized to the cytoplasm in unstimulated cells bound to
an inhibitor I-kB. In response to signal, I-kB is phosphorylated,
ubiquitinated, and degraded in the proteosome. NF-kB translocates
to the nucleus and regulates gene expression.