Download 1 Signal Transduction, II G-proteins, Adenylate Cyclase, Protein

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Signal Transduction, II
G-proteins, Adenylate Cyclase, Protein Kinase A
(readings MBC 734-742, V&V 1268-1269)
Today, we continue our examination of how epinephrine outside of a cell activates
glycogen phosphorylase. Last time we saw that the catecholamine itself stops at the
plasma membrane where it binds to the β-adrenergic receptor.
epinephrine
N
out
cyto
β-adrenergic receptor
C
A conformational change in the receptor is induced by the binding of epinephrine
and this is what transduces information from outside the cell to inside. Where
does the pathway go from here?
I. G-proteins
A. Glucagon and cAMP Glucagon is a hormone that influences carbohydrate
metabolism in the liver. When liver cells are exposed to glucagon, cAMP levels
rise. (cAMP is produced from ATP by the enzyme adenylate cyclase, and we will
talk about it in detail shortly.) Rodbell determined that if liver membranes are
isolated and glucagon is allowed to bind to them, a rise in cAMP can also be seen.
However the amount of cAMP seen would vary widely from a two-fold increase to
many times that. The researchers finally traced this variability to the batches of
ATP that were being used in the experiments. The batches of ATP that gave the
highest cAMP levels were those with a large contaminant of GTP. When the
experiments were done with GTP-free ATP and increasing amounts of GTP were
added, the following curve was seen.
adenylate
cyclase
activity
no glucagon
+ glucagon
(other batch)
+ glucagon
+ glucagon
basal
10-8
10-7
10-6
10-5
[GTP]
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From these data, Rodbell concluded that something must be binding the GTP and
influencing the increase in cAMP caused by glucagon.
B. G-proteins We now know that this something is a G-protein. There are various
kinds of G-proteins known; some are stimulatory (e.g., Gs), others inhibitory (e.g.,
Gi ), and others function in specific sensory pathways (e.g., transducin). All of these
G-proteins are trimeric, consisting of an alpha, beta and gamma subunit.
epinephrine
N
out
cyto
β-adrenergic receptor
γ
C
α
GDP
β
Gs
1. Alpha subunit - alpha chains are about 350 amino acids long. The N-terminus can
be myristoylated (addition of a C14 fatty acid chain) which causes the subunit to
have a high affinity for the membrane, but this is not essential for all alpha
subunits. Sometimes this subunit is also palmitoylated, which would have the
same effect.
Alpha subunits can bind and hydrolyze GTP. This GTPase activity is hormone
dependent. The reaction is: GTP ---> GDP + Pi .
The crystal structure of the alpha chain has been solved. It is composed of two
domains - a helical domain with unclear function and the GTP domain. GTP binds
in the center of the protein and GDP remains stuck in the active site upon
hydrolysis. By comparing the structures of alpha bound to GTP or to GDP, three
regions were identified whose conformation depends on which is bound. So, a
GTP-dependent conformational change is taking place.
2. Beta subunit - beta chains are about 340 amino acids in length. Beta is composed of
seven repeating units of about 42 aa called WD repeats since they contain
tryptophan and aspartate. WD repeats help to form protein-protein interactions
and indeed beta interacts with alpha. From the crystal structure of a trimeric Gprotein, beta is seen to form a strange-looking wheel, rather like an orange with
seven sections. Beta touches two of the regions on alpha that change conformation
in a GTP-dependent manner.
3. Gamma subunit - gamma subunits are about 74 amino acids. Gamma is
isoprenylated at its C-terminus which causes it to associate with the membrane.
The N-termini of both beta and gamma are amphipathic helices which form a
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coiled coil. This keeps beta and gamma permanently associated; the only way to
separate them is to denature them.
C. GTPase activity If the Gs trimer is isolated, GDP will be bound to it. It takes so
long for the GDP to dissociate that it practically doesn’t come off. The rate of
dissociation is 0.01 - 0.001 min-1, so the half-time of dissociation is one to ten hours.
The alpha chain can be engineered into E. coli and produced by itself. If GTP is
added to purified alpha chain, the rate of phosphate formation can be measured.
For the reaction,
rate = 4 min-1
α s-GTP ----> α s-GDP + Pi
This rate is much slower than one would expect from an efficient glycolytic enzyme,
but it is pretty good. However, if one looks at the dissociation of GDP from α s,
rate = 0.3 min-1
α s-GDP ----> α s + GDP
If the beta and gamma subunits are added back to α s-GDP, then the rate of
dissociation slows to the rate we saw above,
rate = 0.01 - 0.001 min-1
α s-GDP + βγ ----> α sβγ + GDP
So, the rate of hydrolysis of GTP by alpha alone is reasonably fast, and if we can get
alpha off of beta-gamma, then the rate of release of GDP can speed up.
D. G-protein cycle (See also Alberts p. 739, Fig. 15-23 and Stryer p. 342 Fig. 13-33.)
R
γ
α
GDP
Ligand
R
α
β
GDP
γ
β
α
GDP
E
Pi
E
RL
γ
β
GDP
α
γ
GTP
β
RL
α
γ
GTP
β
GTP
RL
α
γ
β
Ligand binding to its receptor causes a conformational change in the receptor that
changes how the receptor interacts with the alpha subunit. This changes the
conformation of the α subunit, opening up its active site and allowing GDP to fall
off. GTP binding then induces another conformational change in α that weakens
both the α-β interaction and the α-receptor interaction. The receptor can now
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contact and activate another G-protein trimer. Alpha-GTP can interact with its
effector molecule to activate it. However, α will soon hydrolyze its GTP to GDP, so
GTP hydrolysis is like a clock limiting the time of the active state of α. When
hydrolysis occurs, α-GDP immediately reforms the αβγ complex, which is inactive.
It is not just the freed alpha subunit that can activate downstream effectors.
Sometimes the βγ subunit serves this role and sometimes both α and βγ can carry
information forward.
II. Adenylate cyclase The effector which α s-GTP interacts with in skeletal muscle is
adenylate cyclase which catalyzes the reaction,
ATP ----> cAMP + PPi
where the PPi then gets cleaved to two molecules of inorganic phosphate. The
standard free energy change for the hydrolysis of PPi is -7.5 kcal/mol. But what is
the overall ∆G°’ for the formation of cAMP? It is actually +4.4 kcal/mol. This is
because cAMP is extremely strained. For the reaction,
∆G°’ = -12 kcal/mol
cAMP + H2O ----> AMP
The large release of free energy is due to the relaxing of this strain. Since there is no
free PPi in the cell, adenylate cyclase cannot run backwards.
Adenylate cyclase is a membrane protein with two repeats of 6 transmembrane
stretches connected to a cytoplasmic catalytic domain. (See Alberts p. 737, Fig. 15-21.)
epinephrine
N
Adenylate cyclase
out
cyto
β-adrenergic receptor
γ
C
β
C
N
αs
GTP
ATP
cAMP
It was known since the 1950’s that when skeletal muscle is treated with
catecholamines, its cAMP levels rise. This was the first evidence of second
messengers, of which cAMP is one. Its concentration in a cell can vary by at least 100fold. It’s structure is shown in Alberts p. 736, Fig. 15-19 and Stryer p. 340 Fig. 13-30.
II. Protein Kinase A What effect does an increase in cAMP have? It leads to the
activation of protein kinase A (or PKA, or, in Alberts, cAMP-dependent protein
kinase or A-kinase). PKA is composed of two types of chains - two catalytic
subunits and two regulatory subunits. The form R2C2 is inactive. When cAMP
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levels rise, two molecules of cAMP bind to each R subunit. This changes their
conformation causing them to release the C subunits which are now active. (See
also Alberts p. 740, Fig. 15-24.)
R2C2 inactive
R
+ cAMP
C
C
= cAMP
R
R
C
(active)
R
C
(active)
PKA was actually the first kinase discovered. It was initially considered
unimportant, but now most regulation is known to involve kinases. Kinases are a
class of proteins that transfer the γ-phosphate of an ATP to a substrate.
The target of PKA in skeletal muscle is phosphorylase kinase. Phosphorylase kinase
is an enormous protein, a tetramer of four polypeptides (αβγδ)4. (See schematic of
one such tetramer in Alberts p. 752, Fig. 15-36.) The α and β subunits are each about
130 kD. The γ subunit is the catalytically active subunit and has a structure similar
to that of the PKA C subunit. The δ subunit is calmodulin, a protein we will talk
much more about in the future. PKA phosphorylates first the β and then the α
chains on multiple Ser and Thr residues. The sites where phosphorylation occurs
have the consensus sequences: R R X S/T or R K X X S/T. This phosphorylation
activates the γ chain.
The target of phosphorylase kinase is glycogen phosphorylase itself. (See Alberts p.
741, Fig. 15-25 and Stryer p. 595 Fig. 23-14 A.) Normally phosphorylase (with a MW
of 100,000) is in the form called phosphorylase b, which is a T state with low
activity. The activity of the b form can be raised by high levels of AMP, which is
important for active muscle that is running low of fuel. However, phosphorylase
kinase will phosphorylate glycogen phosphorylase on a single residue (Ser 14)
which converts it to the form called phosphorylase a. Phosphorylase a has a high
activity even at the normal resting levels of AMP.
We still have not seen how the activity of glycogen synthetase is regulated, or how
liver cells, which do not have the β-adrenergic receptor, respond to catecholamines.