Referring back to the last lecture, we learned that there were

two major classes of neurotransmitter receptors, ionotropic

receptors and...
The second major type of neurotransmitter receptor 
 Metabotropic – Neurotransmitter binding causes a
conformational change in the receptor which leads to G
protein binding and to the production of intracellular
metabolites through enzymatic processes. These
responses are of slow onset and long duration,
compared to ionotropic receptors.

These receptors are sometimes referred to as G-protein

coupled receptors (GPCRs) or 7-transmembrane domain
receptors.
 Some history...

In the 1950’s Sutherland and Rall found that stimulation of

cardiac cells with epinephrine resulted in increased
concentrations of a water-soluble nucleotide called
cyclic adenosine monophosphate (cAMP). They proposed
that cAMP acted as a second messenger.

Neurotransmitters, hormones and drugs that cannot cross

the cell membrane (first messengers) exert their effects
inside cells through molecules (second messengers such
as cyclic nucleotides, ions, phospholipids) that act
intracellularly.
Most hormones and many neurotransmitters act by
regulating intracellular second messengers.
In most cases, there are multiple receptors for single
neurotransmitters that bind to GPCRs. This was predicted
from classical binding studies.
For example, there are 5 classes of muscarinic acetylcholine
receptors and 6 classes of adrenergic receptors.
In many cases, the complex pharmacological responses
produced by a single ligand are due to its actions on a
number of different receptors.
The physiological roles of many of these receptors and why
this diversity exists are poorly understood.
 Three components required for G-protein signaling

1. Receptor (on cell surface)

2. G protein (couples to receptor on intracellular side of
cell membrane)
3. Effectors (usually enzymes)

All three are either imbedded in the cell membrane or

tightly associated with it.
 What does a G-protein coupled receptor look like?

Huge family (~ 800 genes)

of related receptors, each a
product of a different gene.
Each contains 7
transmembrane domains.
Neurotransmitter binds to
GPCR on extracellular side
of membrane.
G proteins bind to
intracellular sections of
GPCR.
Neurotransmitter binding
likely stabilizes receptor so
it can efficiently bind the G-
protein trimer.
When a GPCR is stimulated by an agonist, the effect is
usually of limited duration.
There are two major mechanisms limiting the durations of
actions of neurotransmitter agonists at GPCRs:
1. Receptor desensitization - where the extent of interaction
between receptor and G proteins can be decreased.
This is usually accomplished through phosphorylation of
serine & threonine residues on GPCRs, by specific
intracellular enzymes (kinases).
2. Receptor down-regulation - prolonged exposure to
agonist leads to the internalization of receptors from cell
membrane, thus decreasing the numbers of GPCRs that
can interact with G proteins.
 G Proteins

The family of G proteins that transduce signals from

membrane receptors to effector enzymes and ion channels
are known as heterotrimeric (3 different subunits) G proteins.
The three subunits are called ,  and  in decreasing size.
The  subunit binds guanine nucleotides and is the major
mediator of the G protein’s actions on its effector. The  and
 subunits primarily function to support the interactions of the
 subunit with the plasma membrane and with GPCRs, but
like the  subunit, they may also regulate effectors directly.
 The G-protein activation/inactivation cycle
GTP


Agonist

GPCR  GDP


Agonist

GDP GPCR 


Agonist  Pi GTP


GPCR
 
GDP

 The G protein activation/inactivation cycle
In the resting state, the three G protein subunits are bound together with
guanosine diphosphate (GDP) attached to the subunit. This
heterotrimer can bind to an inactive GPCR, or one that has been activated
by an agonist. When an agonist binds to the GPCR, a conformational
change occurs in the receptor, that then leads to a subsequent
conformational change in the G protein associated with it. This
conformational change in the G protein then promotes the unbinding of
GDP from the  subunit of the G protein.
The empty guanyl nucleotide binding site on the  subunit is then
occupied by guanosine triphosphate (GTP) that is present at high
concentrations in cytoplasm. GTP binding causes the subunit to
release from the GPCR as well as dissociate from the  dimer.
The  subunit then activates an effector like adenylyl cyclase or
phospholipase C (the  dimer can also activate effectors like GIRK
channels). Within a few seconds, the intrinsic GTPase activity in the 
subunit hydrolyzes the bound GTP to GDP, inactivating the subunit.
The GDP-bound  subunit dissociates from the effector, re-associates
with thedimer and is ready for another cycle of activation by GPCRs.
Functional diversity of different members of the G protein
family arises from the more than 20 different types of
subunits identified thus far.
Some of the most common are Gs which stimulates
adenylyl cyclase (leading to the production of cAMP) and
Gi, which inhibits adenylyl cyclase.

Some G proteins are widely expressed in cells while others

have a more limited distribution and more specialized
functions. For example, Gta is only found in rods and cones
of the retina.
 Some G proteins are the targets of toxins

The Gs subunit is modified by the cholera toxin such that it

binds ADP-ribose to the guanyl nucleotide binding site. This
prevents the intrinsic GTPase from acting and results in
persistent activation of Gs. In the intestine this leads to
marked elevations in cAMP levels, causing cells to secrete
large amounts of water into the gut, leading to the severe
diarrhea that is seen in cholera infections.
Another bacterial toxin, pertussis, acts on Gi and G0
subunits, preventing their activation by GPCRs. With the
disruption of the inhibitory actions of Gi on adenylyl cyclase,
once again cAMP levels rise, leading to the characteristic
cough seen in whooping cough.
 Effector enzymes regulated by G proteins

Adenylyl cyclases and phospholipases C are the most

common effector enzymes by which G proteins exert their
effects.

Adenylyl cyclases and cAMP as a 2nd messenger

Adenylyl cyclases, imbedded in the cell membrane, catalyze
the synthesis of cAMP from ATP.
There are ten forms of adenylyl cyclase. All are stimulated
by Gs, but differ in their sensitivities to inhibition by Gi.
cAMP acts by activating the cAMP-dependent protein
kinases (protein kinase A; PKA) which can then
phosphorylate other proteins at specific serine or threonine
residues.
 Phospholipase C and phospholipid 2nd messengers

Members of the Gq family of G-proteins transduce signals

between GPCRs and enzymes known as phospholipase C.
These enzymes use phospholipids as substrates.
GPCR occupation by ligand activates Gq. The Gq subunit
binds to the phospholipase on the inner surface of the cell
membrane.
This activated phospholipase then rapidly breaks down the
membrane constituent phosphatidylinositol-biphosphate
(PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG).
IP3 and DAG can both act as 2nd messengers.
IP3 is a small water-soluble molecule that diffuses through
the cytoplasm to bind to specific receptors on the
membrane surrounding the endoplasmic reticulum, resulting
in the release of Ca2+ stores. This in turn initiates a wide
variety of calcium-mediated events in the cell, such as the
activation of calcium/calmodulin-dependent protein kinases.

DAG remains in the cell membrane where, in concert with

phosphatidylserine and calcium, it activates protein kinase
C (PKC), another serine/threonine kinase. Activation of PKC
involves translocating it to the cell membrane where it
becomes able to phosphorylate a wide variety of substrate
proteins such as receptors, ion channels and other
enzymes.
 Signal Amplification

Each GPCR/agonist complex activates several G proteins

before the agonist dissociates.
Each G protein, in turn, initiates the production of many
second messenger molecules.

Thus the binding of agonist to one GPCR can lead to the

activation of many effectors (enzymes).
How do we prove that the action of a drug is through a
G protein coupled receptor...?

1. Intracellular GTP is required.

2. non-hydrolyzable GTP analog greatly prolongs action (or

makes effect irreversible).

3. high affinity GDP--s analog can't be displaced

4. Block with pertussis toxin - inactivates a subset of G

proteins
 GPCR activation
+
G Protein activation

Gs GiG0 Gq

+ - +
Adenylyl cyclase Adenylyl cyclase
Phospholipase C
X
ATP cAMP ATP cAMP PIP2 IP3 + DAG
+
Release of Ca2+ +
PKA from ER + +
+
+
PKC
Protein phosphorylation Ca/calmodulin kinase
+ +
Protein phosphorylation Protein phosphorylation