Signaling into Cells

Part 1: Transmembrane channels and

G-protein linked receptors

Dr. John Wright

School of Medicine
University of Botswana
 Introduction

In Monday’s lecture the general ways in which signal molecules affect

cells were introduced.
The underlying concept that signal molecules stimulate cells with
appropriate receptors was introduced, and we found that binding of a
signal molecule to its receptor activates a set of pre-programmed
responses within the target cell.
We found also that, because signaling is so important in co-ordinating
the physiological behaviour of cells, it has important medical
implications when it goes wrong, and therefore signal processes are
important targets for drug therapy.
In today’s and tomorrow’s lectures we will look at these mechanisms
in more detail, and identify some of the medical implications of the
mechanisms.
 Receptor Classes
Although receptors are very varied it is possible to divide them into
five or six major groups:

1. Receptors which form transmembrane ion channels, opened by

signal binding.
2. Receptors which activate production of a second messenger
within the cell, working via a trimeric G protein.
3. Receptors whose inner domain is an enzyme activated when
the signal molecule binds.
4. Receptors which bind and activate a cytoplasmic enzyme when
the signal molecule binds.
5. Cytoplasmic or nuclear receptors activated by hydrophobic
signal molecules which enter the cell.
6. Cytoplasmic receptors activated by small gas molecules acting
as signals.
 Receptors which are transmembrane ion
channels
Some neurotransmitter receptors are transmembrane ion channels
which open when the neurotransmitter binds.
Only a few of these are known. The best studied example is the acetyl
choline nicotinic receptor (like some other signal molecules acetyl
choline binds to more than one type of receptor carried on different
cells – the other acetyl choline receptor, the muscarinic receptor, works
in a different way).
When acetyl choline binds to the receptor a conformational change
occurs opening a channel through the membrane which can pass
small positively charged ions. This allows sodium ions to flood into the
cell depolarising the membrane and triggering the nervous impulse.
This type of protein is called a ligand gated ion channel.
This type of response can occur very quickly – the nicotinic receptor
can fire about 300 times per second. It is found in nerve endings which
produce a fast response.
 Opening of the Nicotinic Receptor
The nicotinic receptor permits the passage of sodium ions because:
-at its narrowest point it has negatively charged glutamic acid side
chains which attract positive ions
--its narrowest section has hydrophilic surface produced by –OH
groups.
-It is wide enough to allow a hydrated sodium ion through, but is too
narrow to easily allow the slightly bigger potassium ion to pass.

The unactivated (closed) receptor has a ring of large leucine side

chains projecting into the channel, forming a hydrophobic barrier at
the mouth of the narrowest part of the channel.
When acetyl choline binds, the M2 helices twist slightly, swinging the
leucine side chains sideways, and opening the channel.
 Receptors which are ligand gated ion
channels
The acetyl choline receptor forms what is known as a ligand gated
ion channel. Others of the same type are:

Ligand Ion transmitted

Excitatory receptors: acetylcholine Na / K

(nicotinic receptor)
glutamate Na / K
(NMDA receptor)
(non NMDA receptor)
Serotonin (5-OH tryptamine) Na / K
(5HT3 receptor)
Inhibitory receptors: -amino butyric acid Cl
(A class receptor)
Glycine Cl
 Receptors which activate a trimeric G-
protein.

Receptors which activate G-proteins are intrinsic transmembrane

proteins.

When receptors of this type bind their activating messenger (cognate

ligand, in the language of the subject), they change shape, opening a
binding site for a G-protein, which is activated by the receptor.

The activated G protein now activates an effector enzyme, which is

another intrinsic transmembrane protein. This enzyme synthesises an
intracellular messenger molecule (the second messenger) which
carries the message into the cell.

The second messenger is produced from a substrate which is readily

available in the cell.
 The G-Protein Timeswitch

The activated effector enzyme can only remain active for a short time.
The GTPase activity of the G-protein - subunit hydrolyses the bound
GTP to GDP by removing a phosphate.
This inactivates the  subunit, which dissociates away from the effector
enzyme. The effector enzyme is itself inactivated, and the G-protein re-
assembles as an inactive trimer, when the subunit re-attaches to the
 subunits.
Thus the GTPase activity of the - subunit ensures that it acts as a time
switch. It is activated when it exchanges GDP for GTP, but can only
remain in the active form while the GTP remains unhydrolysed. The -
subunit has a low turnover number for GTP – about 1 molecule per
second – but this means that it will turn off after about 1 second’s
activity.
The G-protein may re-activate if the receptor is still occupied after it
switches itself off..
 The G-protein receptor

There are many receptors which can bind to and activate G-proteins,
but all of them are built on the same basic pattern.
They are transmembrane proteins which span the membrane by
means of seven transmembrane helices. They are known as seven
transmembrane helix receptors or serpentine receptors (because
of the way their peptide chain snakes across the membrane).
Loops between the helices form the outer and intracellular portions of
the receptor.
G-proteins always bind to the section formed by the loop between
helices 5 and 6 of the receptor, and the C-terminal tail which follows
helix 7.
The Human Genome Project has discovered many genes coding for
this type of receptor, and it appears to be the commonest protein type
coded in the genome.
 G-protein types, their effectors and
second messengers

There are many types of G-protein, usually defined by the identity of the
 subunit. They can activate a number of different effector enzymes.
Some of the common ones are shown in the table:
G-protein Effector enzyme Second Messenger
Gs Adenylate cyclase cyclic AMP
Gi Phospholipase C Inositol triphosphate +
diacyl glycerol
Gt cGMP phosphodiesterase cyclic GMP
(transducin)
(Light detection in the eye)
Golf adenylate cyclase cyclic AMP
(smell and taste)
Gk potassium channel protein K+
 The Gs Protein

The Gs protein activates adenylate cyclase, which produces cyclic

3’5’AMP from ATP. This acts as a second messenger, transferring the
effects of the extracellular messenger into the cell.
Like the first messenger, cAMP is simply an ‘on’ switch. It must bind to a
receptor/effector which then switches on a pre-programmed response or
set of responses in the cell.
The receptor for cAMP is an enzyme called protein kinase A. This is
normally present in the cell as an inactive tetramer molecule. It has two
catalytic subunits bound to two regulatory subunits. The regulatory
subunits bind to the active site of the catalytic subunits by means of a
pseudosubstrate motif. Binding of cAMP to the regulatory subunit
causes a shape change which removes the pseudosubstrate motif,
releasing the catalytic subunits which are now active.
The catalytic unit now phosphorylates a target protein, bringing about the
cellular effect of the signal.
[cAMP] in the resting cell is usually about 1M. This rises to about 30M
on stimulation. The Kd of protein kinase A for cAMP is about 10M.
 Responses to Cyclic AMP

Though cyclic AMP can only produce one set of responses in any one
cell, in different cell types many different types of response can be
produced via the cAMP system.
1. The activity of an enzyme can be changed. The enzyme will be
activated or inactivated by phosphorylation.
2. The synthesis of a protein can be increased. This will usually be
by activation of gene transcription.
3. The permeability of a membrane can be changed. This will usually
involve the opening of a transmembrane channel. Often this will be an
ion channel, and will stimulate, or more often inhibit, the firing of a
neurone.
4. The release of preformed material may be stimulated. Some
hormone release is`stimulated via a cAMP signal mechanism. The
release of thyroid hormone from granules in response to TSH
stimulation is an example.
 Switching Off the cAMP response

All systems which can be switched on must also have an

‘off’ switch.
Cyclic AMP is broken down to 5’AMP by the enzyme
cAMP dependant phosphodiesterase.
This is a cytoplasmic enzyme. It is not controlled but its
Km is in the same range as the cAMP concentration in
cells (about 1 – 10 M) so its rate rises as the cAMP
concentration increases.
Protein kinase A is inactivated by dissociation of cAMP as
the cAMP concentration falls.
Proteins with phosphorylated serines can be
dephosphorylated by protein phosphatase 1. This may
be controlled in some systems.
 G-protein types, their effectors and
second messengers

There are many types of G-protein, usually defined by the identity of the
 subunit. They can activate a number of different effector enzymes.
Some of the common ones are shown in the table:
G-protein Effector enzyme Second Messenger
Gs Adenylate cyclase cyclic AMP
Gi Phospholipase C Inositol triphosphate +
diacyl glycerol
Gt cGMP phosphodiesterase cyclic GMP
(transducin)
(Light detection in the eye)
Golf adenylate cyclase cyclic AMP
(smell and taste)
Gk potassium channel protein K+
 The Gi Protein System

Gi proteins were first identified because they inhibited adenylate

cyclase (the i stands for inhibitory). However, they are now known to
work by activating a membrane enzyme called phospholipase C.
This enzyme targets a membrane phospholipid, phosphatidyl inositol
4,5 bis phosphate.
It splits the phospholipid head group from this molecule, producing a
water soluble molecule inositol 1,4,5 tri phosphate (IP3), which is
released into the cytoplasm, and leaving behind in the membrane a lipid
soluble molecule, 1,2 diacyl glycerol. Both these molecules can
function as second messengers.
In most systems these two second messengers work synergistically ie.
the response produced by both, working together, is greater than the
sum of the two responses, when activated separately.
The phospholipase C reaction is shown in the next slide.
 IP3 and Ca++ Concentration

Inositol 1,4,5 triphosphate (IP3) is very hydrophilic, so it enters the

cytoplasm, and diffuses to the endplasmic reticulum where it binds to
a calcium channel protein, opening the channel.
The Ca++ concentration in the cytoplasm of an unstimulated cell is
very low – about 10-8M.
The concentration in the endoplasmic reticulum is similar to outside
the cell – about 10-3M. This massive gradient is maintained by ATP
driven pumps.
When a Ca++channel opens through the endoplasmic reticulum
membrane Ca++rapidly floods into the cytoplasm, raising the
concentration to about 10-5M.
Here Ca ++ binds to a detector protein called calmodulin.
Ca++binding proteins other than calmodulin exist in some cells
(troponin C in skeletal muscle is a well known example), but
calmodulin is found almost universally.
 Calmodulin

The receptor protein of the IP3 second messenger is calmodulin,

though it binds Ca++ released in response to IP3, not IP3 itself.
Calmodulin is a small (12 KDa) protein which has four Ca++ binding
sites. It binds Ca++ with a Kd (concentration at which half the binding
sites are occupied) of about 10-6M.
Binding of Ca++ produces a major shape change in calmodulin which
can be transmitted to produce shape changes in proteins with which
it is associated.
Calmodulin is found in association with many protein systems, for
example, myosin head groups in smooth muscle, where it controls
actin – myosin interaction in response to Ca++ signalling.
The closely related protein troponin C has a similar role in the
myosin head groups of striated muscle.
 1,2 Diacyl Glycerol

Although IP3 was initially recognised as the second messenger of

the Gi protein system, 1,2 diacylglycerol is now also recognised as
an equally important second messenger.
1,2 DAG activates an enzyme called protein kinase C. This is an
inactive cytoplasmic enzyme in unstimulated cells but binding of Ca ++
causes it to bind to membrane phosphatidyl serine.
Once attached to the membrane it can come into contact with 1,2
diacylglycerol, which activates it, by pulling a pseudosubstrate
domain away from the serine kinase active site of the enzyme
(compare this to the mechanism of activation of protein kinase A
which similarly involves removal of a pseudosubstrate).
Protein kinase C can phosphorylate many target proteins in different
cells but commonly phosphorylatyes and activates transcription
factors, promoting cell growth and diffentiation.
 Synergistic Action of IP3 and 1,2 DAG

Experimentaly it is possible to simulate the IP3 stimulation without 1,2

DAG stimulation, by using a Ca++ ionophore – a molecule which fits into
the plasma membrane providing a channel for Ca++ ions.
It is also possible to stimulate protein kinase C directly without IP3
stimulation using molecules called phorbol esters.

In most cells simulation of the action of just one second messenger (IP3
or 1,2 DAG) produces a relatively weak response – nothing like as big
as the full physiological response.

Stimulation by Ca++ionophore and phorbol ester together produces a full

physiological response.
 Light Detection in the Eye

Surprisingly, light detection by the visual pigments works by a G

protein mechanism, involving a G-protein called Gt or transducin.
The visual pigment in the rod cells of the retina is called
rhodopsin. It consists of a seven transmembrane helix type
protein called opsin, to which is bound a vitamin A aldehyde co-
factor called retinal.
Retinal is only able to bind the opsin molecule because one of
several double bonds in the 15C long tail of the molecule (at
carbon 11) is in the cis configuration, putting a bend in the
molecule which allows it to fit a binding site on opsin.This 11-cis
retinal /opsin molecule forms rhodopsin.
Rhodopsin is able to absorb light with a max of 590nm.
However it is light sensitive – when it absorbs light the 11-cis
retinal is converted to the straight chain all trans configuration.
This is no longer able to bind to the opsin and comes away from
the binding site. This change in structure of rhodopsin bleaches
the molecule – its max changes to 320nm in the uv region.
 How ‘bleached ‘ rhodopsin passes on the
visual message.
When 11 cis retinal is converted to all trans retinal and can no longer bind
to opsin, the opsin molecule undergoes a conformational change.
This allows it to bind a trimeric G-protein, Gt , dissociating it’s  sub unit
from its  sub units. The  sub unit is the able to activate a
transmembrane protein cyclic GMP phosphodiesterase.
This protein breaks down a signal molecule, cyclic GMP, to inactive
5’GMP.
Cyclic GMP is normally present in the rod cell. It is produced by an active
guanyl cyclase, and it holds open a Na+ channel through the membrane.
This normally allows sodium ions to leak through the membrane. They are
pumped in the opposite direction by an ATP driven sodium ion pump.
Between them these two proteins set up a ‘dark current’ of sodium ions
across the membrane. This is interrupted when cGMP is removed by the
phosphodiesterase activated by light.
The resulting hyperpolarisation triggers the rod cell response.