Download Document

Lecture 2, Oct 11 Important points from 10/7:
Ligands and Receptors
Ligand-receptor binding shows great sensitivity.
Endocrine, paracrine, autocrine, membrane-bound
Hydrophilic ligands bind to cell surface receptors
Cell surface receptors: G protein coupled; ion-channel linked;
receptor tyrosine kinase linked; receptors with intrinsic
enzymatic activity
Second messengers: inside the cell—effector molecules of cell
signaling
Signaling: protein kinases; GTP-binding proteins with GTPase
activity can function as molecular switches; integration of
multiple signals; internalization; protein-protein interactions using
adapter proteins.
Signal Transduction: focus on G-proteins and the PKA pathway
Many cell-surface receptors are coupled to trimeric
signal-transducing G proteins.
Trimeric: composed of three different subunits
G proteins: bind either GTP or GDP
Ligand binding to a G protein coupled receptor activates
the associated G protein which in turn activates an
effector enzyme to generate an intracellular second
message.
All G-protein coupled receptors have 7 membrane spanning regions
with their amino termini on the extracellular face and their carboxy
termini on the cytoplasmic face of the plasma membrane.
Structure of an inactive G protein—alpha and gamma covalently
attached lipid molecules; alpha is GDP bound.
Structure of transducin, the G protein in visual transduction
Taste receptors
Mu opioid receptor
Ion channel on pain fibers
Illustration: binding of epinephrine/norepinephrine
to the b adrenergic receptor
Mediates the body’s response to stress (e.g. going to the dentist)
--release of glucose and fatty acids from liver and fat cells
--increased contraction of cardiac muscle
Binding of epinephrine to b adrenergic receptor increases the
intracellular concentration of cAMP. How does this happen?
cAMP is synthesized within
cells from ATP by the enzyme
adenylate cyclase.
cAMP is degraded by the
enzyme cAMP
phosphodiesterase
--The b-adrenergic receptor
mediates the induction of
epinephrine-initiated cAMP
synthesis.
--Different receptors utilize a
common adenylate cyclase (i.e.,
each receptor does not have its
own intrinsic adenylate cyclase).
Martin Rodbell, Nobel Prize 1994
GTP is required for the ligand-induced
stimulation of adenylate cyclase.
Overall: need, 1) a receptor, 2) a
transducer (G-protein) and 3) an
amplifier (adenylate cyclase) that
generates large amounts of a second
messenger.
--Binding of the ligand to the
receptor changes its conformation,
causing it to bind to the trimeric Gs
protein in such a way that GDP is
displaced from Gas and GTP is
bound.
--The Gas-GTP complex
dissociates from the Gbg complex,
then binds to and activates
adenylate cyclase.
--activation is short-lived: GTP
bound to Gas hydrolyzes to GDP in
second, leading to the association of
Gas with Gbg and inactivation of
adenylate cyclase.
Disassembly of activated
G-protein produces TWO
signaling components.
Switching off of the G-protein alpha subunit by hydrolysis of its bound GTP
Cholera toxin: modifies
Gas by adding an ADP
ribosyl group. This
modified Gas can bind
GTP but cannot hydrolyze
it. As a result, there is an
excessive and
nonregulated rise in
intracellular [cAMP].
Activating mutations in Gas underlie fibrous dysplasia,
where see excess fibrous growth, which calcifies over time
Note: inactivating mutations in Gas underlie another
disease, pseudohypoparathyroidism
To review: G protein activation and
complex formation are part of a cycle
The Gas stimulates adenylate cyclase; another Ga
subunit, Gai, inhibits adenylate cyclase.
Degradation of cAMP
is also regulated
through the hydrolysis
of cAMP to 5”-AMP by
cAMP
phosphodiesterase.
Many drugs affect
cAMP
phosphodiesterase—
including caffeine.
How does increased cAMP activate the cAMPdependent protein kinase, PKA?
The catalytic subunit of PKA can phosphorylate
substrates on serine or threonine residues. It has
substrates in the cytoplasm and the nucleus.
In the nucleus, PKA can activate transcription of
genes containing cAMP response elements, or CREs
in their promoter. A specific transcription factor, the
cAMP response element binding protein, CREB,
binds to this sequence and activates transcription of
downstream genes. When CREB is
unphosphorylated, it is inactive; only in its
phosphorylated state does CREB activate
transcription.
How ligand binding to a cell surface receptor can induce gene
expression
How ligand binding to a cell surface receptor can induce gene
expression
Animation: see http://www.whfreeman.com/lodish/
Lodish book, 5th edition, chapter 14, animation on
extracellular signaling.
Or Alberts et al., 4th edition, interactive disk
Important points:
•G protein coupled receptors: receptors with 7 membrane
spanning domains.
•Ligand binding produces signaling to second messenger by
binding to and transducing its signal to a trimeric G protein
•G protein has 3 subunits: a, b and g. Ligand-bound receptor
interacts with G protein, causing conformational change. Ga
subunit exchanges GDP for GTP and dissociates from Gbg. Both
a and b/g can be active signaling components.
•GTP-bound Ga subunit now associates with and activates
adenylate cyclase, which produces cAMP, a second messenger.
Gas activates adenylate cyclase, Gai inhibits adenylate cyclase.
•Intrinsic GTPase activity of Ga terminates signaling.
•cAMP activates PKA by binding to the regulatory subunits of
the kinase. PKA is a tetrameric kinase: 2 regulatory and 2
catalytic subunits. When cAMP binds the regulatory subunits,
the catalytic subunits translocate into the nucleus where they can
phosphorylate substrates. These can include transcription factors
such as CREB and thereby result in changes in gene expression.