Second year college lecture on GPCRs.ppt

2nd year student lecture on GPCRs

1. Cell Structure &
Function GPCRs

2. Lecture 7&8 learning objectives
Advanced GPCRs
•Describe GPCR structure, G proteins, mechanisms
of action.
•Understand the role of GTPases and other enzymes
•Disruption of GPCRs by disease or infection

3. Which picture is a GPCR?
A B C

4. Three Largest Classes of Cell-Surface
Receptor Proteins
Ion-Channel-linked, G-Protein-linked, and
Enzyme-linked Receptors
•Three classes - defined by the
transduction mechanism they use.
•Cell-surface receptor proteins act
as signal transducers.
•They convert an extracellular
ligand-binding event into
intracellular signals that alter the
behaviour of the target cell
(response)

5. Why an advanced lecture on GPCRs?
•The human genome encodes nearly 800 GPCRs, representing over 3% of
human genes..
•GPCRs recognize a wide variety of signals ranging from photons to ions,
proteins, neurotransmitters, and hormones.
•Impaired ligand concentration, GPCR protein expression, or mutation and
signaling are implicated in many pathophysiological conditions, including
central nervous system (CNS) disorders, cardiovascular and metabolic diseases,
respiratory malfunctions, gastrointestinal disorders, immune diseases, cancer,
musculoskeletal pathologies, and eye diseases.
•Targeting of GPCRs is hence widely utilized for therapeutic intervention; GPCRs
correspond to 30% of all identified drug targets and remain major targets for
new drug development. In fact, researchers estimate that between one-third
and one-half of all marketed drugs act by binding to GPCRs.

6. The GLP-1 receptor, or Glucagon-like peptide-1 receptor, is a G protein-
coupled receptor (GPCR).
The GLP-1 receptor specifically binds to glucagon-like peptide-1, a
hormone that regulates blood sugar levels and plays a significant role
in glucose homeostasis and the secretion of insulin.
Activation of the GLP-1 receptor by GLP-1 or related molecules can lead
to increased insulin secretion, reduced glucagon secretion, and
improved blood sugar control, making it an important target for the
treatment of diabetes.

7. Recap on high level GPCRs MOA
•The signal molecules that activate them are
as varied in structure as they are in function:
includes proteins and small peptides, as well
as derivatives of amino acids and fatty acids.
•The same ligand can activate many different
receptor family members; eg. at least 9
distinct G-protein-linked receptors are
activated by adrenaline.
•When extracellular signaling molecules bind
to serpentine receptors, the receptors
undergo a conformational change that
enables them to activate trimeric GTP-
binding proteins (G proteins).
•These G proteins are attached to the
cytoplasmic face of the plasma membrane,
where they serve as relay molecules,
functionally coupling the receptors to
enzymes or ion channels in this membrane.
•There are various types of G proteins, each
specific for a particular set of serpentine
receptors and for a particular set of
downstream target proteins in the plasma
membrane.
All have a similar structure and operate in a
similar way.

8. G protein linked receptors
•Despite the chemical and functional
diversity of the signal molecules that
bind to them, all G-protein-linked
receptors have a similar structure.
•They consist of a single polypeptide
chain that threads back and forth
across the lipid bilayer seven times
and are therefore sometimes called
serpentine receptors
•In addition to their characteristic
orientation in the plasma membrane,
they have the same functional
relationship to the G proteins they
use to signal the cell interior that an
extracellular ligand is present.

9. GPCR MOA (revision slide)
1.Ligand (wide variety, hormones,
proteins, neurotransmitters, ions,
photons).
2.Receptor (7 transmembrane)

10. G proteins
•G proteins are composed of three protein subunits , , and . In the unstimulated state, the  subunit has GDP bound and the G protein is inactive
•GTP binding causes conformational change that affects the surface of the  subunit that associates with the  complex in the trimer. This change causes (1) release of the  complex and (2) causes and the  subunit to adopt a new shape that allows it to interact with its target proteins.
•The  complex does not change its conformation, but the surface previously masked by the  subunit is now available to interact with a second set of target proteins.
• The targets of the dissociated
components of the G protein
are either enzymes or ion
channels in the plasma
membrane, and they relay the
signal onward.

11. Quick Question…
•Looking at the G protein structure in (B) below, why do you think they named the green part alpha and the blue beta?

12. G proteins
•When stimulated by an
activated receptor, the
 subunit releases its
bound GDP, allowing
GTP to bind in its
place. This exchange
causes the trimer to
dissociate into two
activated components
an  subunit and a 
complex

13. GPCR MOA (revision slide)
1.Ligand (wide variety, hormones,
proteins, neurotransmitters, ions,
photons).
2.Receptor (7 transmembrane)
3.G Protein (alpha, beta, gamma)

14. G proteins
•The  subunit is a
GTPase, and once it
hydrolyzes its bound
GTP to GDP, it re-
associates with a 
complex to re-form
an inactive G protein,
reversing the
activation process

15. GTPase
•GTPases are a large family of hydrolase enzymes that bind to
nucleotide guanosine triphosphate (GTP) and hydrolyze it to
guanosine diphosphate (GDP).
•The time during which the  subunit and  complex remain apart
and active is usually short, and it depends on how quickly the 
subunit hydrolyzes its bound GTP.
•An isolated  subunit is an inefficient GTPase, and, left to its own
devices, the subunit would inactivate only after several minutes.
• Its activation is usually reversed much faster than this, however,
because the GTPase activity of the  subunit is greatly enhanced by
the binding of a second protein, which can be either its target
protein or a specific modulator known as a regulator of G protein
signaling (RGS) - crucial role in shutting off G-protein-mediated
responses in all eucaryotes.

16. GEF & GAP
•Guanine nucleotide exchange factors (GEFs) and GTPase-
activating proteins (GAPs) regulate the activity of small guanine
nucleotide-binding (G) proteins to control cellular functions. In
general, GEFs turn on signaling by catalyzing the exchange from
G-protein-bound GDP to GTP, whereas GAPs terminate signaling
by inducing GTP hydrolysis. GEFs and GAPs are multidomain
proteins that are regulated by extracellular signals and localized
cues that control cellular events in time and space. Recent
evidence suggests that these proteins may be potential
therapeutic targets for developing drugs to treat various
diseases, including cancer.

17. GPCR MOA (revision slide)
1.Ligand (wide variety, hormones, proteins,
neurotransmitters, ions, photons).
2.Receptor (7 transmembrane)
3.G Protein (alpha, beta, gamma)
4.GTPase (GDP to GTP) and conformational
change in G subunits

18. Cyclic AMP is a second messenger used
by one class of G proteins
•Cyclic AMP is synthesized from ATP by a plasma-
membrane-bound enzyme adenylyl cyclase
•Then rapidly and continuously destroyed by one or more
cyclic AMP phosphodiesterases that hydrolyze cyclic
AMP to adenosine 5 -monophosphate (5 -AMP)

19. Adenylyl Cyclase
•Adenylyl cyclase is a signal amplifier.
•It acts at the center of the signaling cascade
that transduces binding of hormones into
cellular responses. When hormones such as
adrenaline or glucagon bind to G-protein
coupled receptors, they activate G-proteins,
which in turn activate adenylyl cyclase.
•Adenylyl cyclase then performs its catalytic
reaction, clipping off two phosphates from
ATP and forming an additional bond to the
remaining phosphate. The resultant
molecule, cyclic AMP or cAMP, is released
and travels quickly throughout the cell,
regulating the function of multiple proteins.
•In this role, cAMP is often called a "second
messenger", delivering the original message
of the hormone. As an added benefit, the
signal is amplified in the process, because
adenylyl cyclase is an enzyme that can create
many molecules of cAMP when activated.

20. Amplification of Adrenaline Signal

21. An increase in cyclic AMP in response to an extracellular signal.
This nerve cell in culture is responding to the neurotransmitter serotonin, which acts
through a G-protein-linked receptor to cause a rapid rise in the intracellular
concentration of cyclic AMP. To monitor the cyclic AMP level, the cell has been
loaded with a fluorescent protein that changes its fluorescence when it binds cyclic
AMP. Blue indicates a low level of cyclic AMP, yellow an intermediate level, and
red a high level. (A) In the resting cell, the cyclic AMP level is about 5 × 10
-8
M. (B)
Twenty seconds after the addition of serotonin to the culture medium, the
intracellular level of cyclic AMP has increased to more than 10
-6
M, an increase of
more than twentyfold.

22. GPCR MOA (revision slide)
1.Ligand (wide variety, hormones,
proteins, neurotransmitters, ions,
photons).
2.Receptor (7 transmembrane)
3.G Protein (alpha, beta, gamma)
4.GTPase (GDP to GTP) and
conformational change in G subunits
5.Cyclic AMP (adenylyl cyclase and
cyclic AMP phosphodiesterases)

23. Actions of cAMP
•Skeletal muscle and liver cells – breakdown of
glycogen
•Cardiac muscle – strengthens heart contraction
•Smooth muscle – inhibits contraction
•Blood platelets – inhibits their mobilization during
blood clotting
•Intestinal epithelial cells – secretions of salts and
water into the gut
•EACH CELL HAS PREPROGRAMMED RESPONSE TO
cAMP

24. Cyclic-AMP-dependent Protein Kinase
(PKA) Mediates Most of the Effects of
Cyclic AMP
•PKA catalyzes the transfer of the terminal phosphate group from ATP to
specific serines or threonines of selected target proteins, thereby
regulating their activity - Phosphorylation.
•PKA is found in all animal cells and is thought to account for the effects
of cyclic AMP in most of these cells. The substrates for PKA differ in
different cell types, which explains why the effects of cyclic AMP vary so
markedly depending on the cell type.
•In the inactive state, PKA consists of a complex of two catalytic subunits
and two regulatory subunits. The binding of cyclic AMP to the regulatory
subunits alters their conformation, causing them to dissociate from the
complex. The released catalytic subunits are thereby activated to
phosphorylate specific substrate protein molecules

25. GPCR MOA (revision slide)
1.Ligand (wide variety, hormones,
proteins, neurotransmitters, ions,
photons).
2.Receptor (7 transmembrane)
3.G Protein (alpha, beta, gamma)
4.GTPase (GDP to GTP) and
conformational change in G subunits
5.Cyclic AMP (adenylyl cyclase and
cyclic AMP phosphodiesterases)
6.PKA enzyme (phosphorylates it
substrate)

26. How gene
transcription is
activated by a rise in
cyclic AMP
concentration
Once activated,
PKA phosphorylates its
substrates, such as CREB, Raf,
Bad and GSK3, and then
regulates gene expression, cell
survival and migration

27. GPCR MOA (revision slide)
1.Ligand (wide variety, hormones,
proteins, neurotransmitters, ions,
photons).
2.Receptor (7 transmembrane)
3.G Protein (alpha, beta, gamma)
4.GTPase (GDP to GTP) and
conformational change in G subunits
5.Cyclic AMP (adenylyl cyclase and
cyclic AMP phosphodiesterases)
6.PKA enzyme (phosphorylates it
substrate)
7.CREB (regulates gene expression)