Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5843_Библиотеки_им_академика_М_И_Перельмана.pdf
92 ESSENTIAL PHARMACOLOGY
Receptors for molecules that cannot readily cross the cell mem-
brane because they are charged, such as the biogenic amines and
peptides, must be on the surface of the cell, typically in a trans-
membrane orientation so that they can transduce signals across
the membrane. Receptors for molecules that are hydrophobic and
can diffuse across the lipid bilayer, such as steroid hormones, vita-
mins A and D, or have specific transport proteins, as does thyroid
hormone, are usually in the cytosol or the nucleus of the cell, and
their ultimate site of action is the DNA, where they act as tran-
scription factors to modulate gene expression. These locations
obviously influence the nature of the drugs we can design to inter-
act with the receptors: Hydrophobic drugs are needed to approach
the intracellular receptors, while permeability is not as important
an issue for drugs that can attach to the extracellular portion of
receptors.
Ligands that bind receptors on the cell surface are not trans-
ported into the cell to carry out their signaling function. Rather,
they stimulate the receptor protein to carry out events that gen-
erate a new signaling molecule in the cell – a so- called “second
messenger.” It appears that there is a much smaller number of
intracellular second messengers than there are extracellular “first
messengers.” The intracellular “vocabulary” is smaller. The best-
known second messengers are the calcium ion and cyclic nucleo-
tides (cyclic AMP and cyclic GMP). These second messengers act
by binding to and activating their own targets inside the cell, such
as calcium- binding proteins or protein kinases regulated by cyclic
nucleotides.
In addition to these classical second messengers, activation of
some receptors can lead to the hydrolysis of phosphatidylinositol
Biological Receptor- Effector Mechanisms 93
bisphosphate on the inner surface of the cell membrane. Break-
down of this phospholipid releases two second messengers: ino-
sitol trisphosphate (IP3) and diacylglycerol. The release of IP3
serves as another way of introducing calcium into the cytoplasm of
the cell because it binds to a target protein on the membrane of the
endoplasmic reticulum that is a calcium channel. This IP3 recep-
tor responds to binding IP3 by acting as a channel to allow calcium
that has been stored in the endoplasmic reticulum to flow into the
cytoplasm. The diacylglycerol, in a manner analogous to the cyclic
nucleotides, activates a protein kinase, protein kinase C. Calcium
that is released by the action of IP3 also stimulates protein kinase
C, so both of these signaling molecules, calcium and diacylglycerol,
can act in a synergistic manner.
We can broaden the concept of “second messengers” if we think
of the information flow that can be carried by networks of protein
kinases and of transcription factors. Some cell surface receptors are
ligand- activated protein kinases. When they bind an agonist, they
phosphorylate a protein inside the cell. In many cases this target
protein is itself a kinase, which can then phosphorylate other pro-
teins. Chains of kinases may be organized in this fashion three or
four layers deep, leading in some cases to a kinase that enters the
nucleus and influences gene expression by phosphorylating tran-
scription factors.
Information also flows through networks of transcription fac-
tors. Nuclear receptors for lipid- soluble hormones like the steroid
hormones estrogen and testosterone promote the expression of
specific genes when they are activated by agonists. However, often
the genes controlled encode transcription factors themselves– both
activators and inhibitors of transcription of other genes. Thus, very
complex gene expression networks can be activated, transferring
information to sets of genes.
94 ESSENTIAL PHARMACOLOGY
The organization of biological signaling into tiers of first and sec-
ond messengers has important consequences for pharmacology.
Since the number of second messenger species is much smaller
than that of first messengers, drugs and endogenous agonists can
often be classified into groups depending upon which second mes-
senger systems they influence. What can seem a bewildering array
of drugs can thus be greatly simplified. If one knows that a certain
drug increases calcium in a cell, reduces cyclic AMP, or activates
a “master” kinase, then one can easily relate its action to that of
other drugs, hormones, or neurotransmitters. On the other hand,
a limitation to pharmacology is that sometimes a new drug, found
to act on a newly discovered receptor, is found to do nothing more
than a well- known drug because it is modifying the same second
messenger system in the cell.
The structure of receptors often reflects their roles as
transducers of information from a first to a second messen-
ger (see Figure 10.1 for examples). Typically there must be a
domain that has a binding pocket for the first messenger, and,
if the receptor is in the plasma membrane, this domain must be
oriented toward the outside of the cell. Then there is usually a
second domain that is involved in second messenger generation.
For example, the insulin receptor has a protein kinase domain
on the cytoplasmic side of the membrane that initiates a phos-
phorylation cascade. The atrial natriuretic peptide receptor has
a guanylate cyclase domain that generates the second messen-
ger cyclic GMP. The nicotinic acetylcholine receptor is a complex
of several homologous, transmembrane subunits that together
Biological Receptor- Effector Mechanisms 95
form an ion channel through the membrane. Agonist activation
opens this channel to allow calcium to enter the cell as a sec-
ond messenger, along with sodium. Entry of both of these cations
leads to depolarization of the membrane, which activates other
voltage- dependent calcium channels in the membrane, further
amplifying the calcium signal. The estrogen receptor has a dis-
tinct domain that binds DNA at sequence- specific locations and
promotes gene transcription.
Extracellular
space
Agonist-gated
ion channel
Agonist-regulated
enzymes
G protein-coupled
receptor
Plasma
membrane
LB LB
Ions
Pro Kin
Pro
Pro-PO
4
GTP
Gua Cyc
Cyclic
GMP
G
Transcriptional
regulator
LB
DNA
Bi
Nucleus
Cytoplasm
Structural motifs and locations of representative physiological
receptors that act as protein kinases (Pro Kin) or guanylate cyclases (Gua
In contrast, the nuclear receptors, which regulate the transcription of DNA, are
th
96 ESSENTIAL PHARMACOLOGY
There is a major class of receptors that must interact with other
proteins to lead to second messenger generation. These are the
so- called G- protein coupled receptors, and they deserve detailed
description because of their large number and importance in a
wide range of signaling processes. They may also be some of the
first receptors to have evolved. They are transmembrane proteins
formed by a complex of 7 alpha helices bundled together. This motif
appeared hundreds of millions of years ago in certain bacteria that
harvest sunlight. In this case, the protein, called bacteriorhodopsin,
has a small light- absorbing compound, a retinal, covalently bound
amongst the helices. When a photon of light is absorbed by this
compound, the energy is harvested and used to establish ion gradi-
ents across the cell membrane. This signal reception device is now
used in our own retinas – not to harvest energy, but to generate a
signal indicating the presence of light. More generally, proteins of
this design have empty pockets where the retinal is in rhodopsin, and
this pocket is the binding site for signaling ligands. We have literally
hundreds ofdistinct receptors in this class lining our olfactory epithe-
lium. Each such receptor is designed to be activated by different odor-
ant molecules, sending signals through nerve activation to our brain.
These are striking examples of the use of G- protein coupled
receptors to sense our environment. It seems natural then that
cells and organs in our bodies have adopted these same signaling
machines to sense signals from one another within the confines
of the body.
The 7 transmembrane domain receptors influence second messen-
ger generation in cells through activation of G- proteins on the intra-
cellular face of the membrane. The G- proteins comprise a family of
transduction proteins that activate or inhibit processes involved in
the formation of second messengers. The G- protein is a trimer of
Biological Receptor- Effector Mechanisms 97
three subunits: an alpha subunit of mass about 39 kDa, a beta subu-
nit of mass about 35 kDa, and a gamma subunit of mass 11 kDa.
The activation/inactivation cycle of the G- protein follows a series
of steps illustrated in Figure10.2.
1. In the absence of an agonist, the receptor is in a “resting” state
R, as is the G- protein. In this state the G- protein has GDP bound
to the alpha subunit.
2. Binding of an agonist to the receptor then causes (or stabilizes)
a conformational change of the receptor (R*) that favors inter-
action with the G- protein. This interaction in turn causes the
G- protein to release GDP and bind GTP in an exchange reaction.
The binding of GTP holds the alpha subunit in an active confor-
mation even as it diffuses away from the receptor and dissociates
from the beta and gamma subunits.
P
i
G−GTPG−GDP
GDP
GTP
Cell membrane
E*
R*
Agonist
R
E
th
edition, 2023, Figure 2–10 /
98 ESSENTIAL PHARMACOLOGY
3. The alpha subunit can then interact with effector proteins (E),
such as the enzyme adenylate cyclase that generates the sec-
ond messenger cyclicAMP. The consequence of this interaction
depends on the type of G- protein involved. The Gs- protein stim-
ulates the cyclase, enhancing cAMP production, while the Gi-
protein inhibits the cyclase.
4. The time period during which the G alpha subunit remains
active is limited. The alpha subunit carries its own timer, in a
sense, as it is a GTPase that can hydrolyze the GTP to GDP so
that the protein returns to its relaxed conformation. This GTPase
activity is low, so the G protein remains active for several min-
utes. However, when the alpha subunit interacts with an effec-
tor, the interaction enhances the GTPase activity. This ability to
activate the GTPase is referred to as “GAP” activity, for GTPase
Activating Protein, and represents a form of negative feedback
to reduce signaling through the G- protein pathway. Additional
proteins that have “GAP” activity have been more recently dis-
covered, revealing additional control pathways that may regulate
G- protein signaling.
5. As soon as the bound nucleotide has been reduced to the GDP
form, the alpha subunit can no longer activate effectors, and it
recombines with the beta- gamma G protein subunit dimer.
Initially it was thought that the beta- gamma subunit dimer
acted only as a buffer in this system to sequester inactive alpha
subunits. However, it is now known that the beta- gamma dimer
can also bind to and influence the activity of certain effectors.
As an example, one important effector in cardiac muscle is a
potassium channel. When acetylcholine from the vagus nerve is
released on muscarinic receptors on the heart muscle, the acti-
vation of a G- protein by the receptor causes the release of beta-
gamma dimers that activate the potassium channel. The duration
of the influence of the beta- gamma dimer is apparently limited
by the same clock as the alpha subunit– hydrolysis of the GTP
on the alpha subunit allows the alpha subunit to recapture and
inactivate the beta– gamma dimer.
Biological Receptor- Effector Mechanisms 99
Amplification of signals. The strength of a signal coming into
a cell can be amplified by G- proteins in the sense that a single
receptor molecule can activate several G- protein molecules while
it is in an activated state. These activated G- proteins can then
activate several effector molecules before they decay through
hydrolysis of bound GTP. The overall effect is that a single agonist
molecule can activate multiple copies of an effector enzyme like
adenylate cyclase.
Diffusion of signals. A signal brought to a cell by a particular
agonist can diffuse into multiple signaling pathways through the
G- protein network. This is because a single type of receptor mol-
ecule can activate several different types of G- protein, such as Gs
and Gq. These different types of G- proteins can activate different
types of effectors; in fact, a single G- protein, like Gs, can activate
different types of effectors through both the alpha subunit and the
beta- gamma dimer. The net result is the activation (or inhibition) of
a broad array of effector molecules.
Integration of signals. The G- protein network can also inte-
grate signals coming from different pathways. Two different ago-
nists may bind to two different receptors, but both receptors may
interact with the same class of G proteins. Thus the incoming sig-
nals from the two pathways can be summed to give a larger net
signal than would arise from either pathway alone. Alternatively,
one agonist could lead to activation of a G- protein that activates an
effector, just as Gs activates adenylate cyclase, while another ago-
nist could activate the inhibitory G- protein, Gi. The net activation
of the cyclase would be the sum of the positive and negative signals
coming through these two pathways.
This interplay among signaling pathways mediated by
G- proteins is obviously important for the pharmacologist or clini-
cian to consider when trying to predict or understand the effects of
drugs that alter one component of such networks.
100 ESSENTIAL PHARMACOLOGY
Signaling systems in the body are continually adjusting amplifica-
tion and feedback mechanisms to keep biochemical signals in the
range in which our cells and organs can respond. Many of these
adjustments occur at the level of receptors, which are modified, cre-
ated, or destroyed in reaction to the ligand concentrations to which
they are exposed.
The beta adrenergic receptor provides a fairly well- understood
example of how these “gain” adjustments (to use a term from elec-
trical engineering for signal amplification) are made in vivo (see
Figure10.3). In the absence of an agonist, such as norepinephrine,
the receptor is in a “resting” state with a characteristic high affin-
ity for the agonist. When the agonist binds, the receptor interacts
with a G- protein, say Gs, a stimulatory G- protein, promoting the
release of GDP and the loading of GTP onto the G- protein. This
leads to immediate negative feedback: The new conformation of
the G- protein encourages the receptor to change its conformation
to one of lower affinity for the agonist.
Thus, the agonist falls off more readily, terminating the signal.
As long as the receptor remains in an activated state, it is also
in a conformation that is attractive to a patrolling enzyme, a pro-
tein kinase called BARK, for Beta Adrenergic Receptor Kinase (or
more generally, GRK, for G- protein Receptor Kinase), that detects
activated receptors and phosphorylates them. When the receptor is
phosphorylated, it is recognized by another patrolling protein, beta
arrestin, that binds to the phosphorylated receptor and “arrests” it
by blocking its ability to activate G- proteins.
If the receptor remains bound to beta arrestin for a significant
period of time, the complex of the receptor and beta arrestin is rec-
ognized by the “assembly protein” AP2 that initiates the assembly
of a coat of clathrin molecules on the portion of the membrane in
which the receptor is embedded. This coat then invaginates, forming
a clathrin- coated pit, which then buds off of the plasma membrane
Biological Receptor- Effector Mechanisms 101
and carries the receptor into the interior of the cell, where it can no
longer be activated by the agonist.
These sequestered receptors may be spared and returned to the
cell surface if the signal strength declines, but if not, they are carted
away to the cell’s lysosomes and destroyed by hydrolytic enzymes.
Each feedback step in this signal pathway is reversible so that in
the absence of ligand, the system is “reset” so it can respond again.
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
21
GRK
ATP
5
G
s
P
P
P
P
P
β
-AIT
P
4
3
6
Pase
Endosomes
Lysosome
Coated pit
Agonist in extracellular space
-
surface (steps 4 and 5) for re- use.
th
edition, 2023, Figure 2–12 /