G-protein-linked receptors

Brian Pierchala
BIO402/502
MBOC4 Chapter 15
G-protein-linked receptors
**These are also calld G-protein-coupled receptors (GPCRs).
**GPCRs comprise the largest family of cell surface receptors. In mice, there are 1000 different
receptors involved in smell alone.
**A single ligand often binds to more than one GPCR. The neurotransmitter serotonin, for
example, has at least 15 GPCRs.
**Half of the known medications we take work through GPCRs.
**All GPCRs have the characteristic 7 transmembrane domains with the same topology: the N-
terminus is extracellular and typically comprises most of the ligand binding domain. The C-
terminus is intracellular.
**All GPCRs signal via the use of G-proteins.
Inactive heterotrimeric G-protein
**Heterotrimeric G-proteins are tethered to the membrane via lipid anchors. Both the alpha and
gamma subunits have covalent lipid modifications.
**In an inactive state, the alpha subunit binds GDP and is a GTPase. The beta subunit, which
binds to the alpha subunit, locks the alpha subunit in an inactive conformation.
**There are several types of G-proteins, each of which bind to a particular set of GPCRs, and to
a particular set of intracellular signaling molecules.
G-protein activation

1. When not activated, the alpha subunit binds GDP.

2. When the ligand binds to the GPCR it changes
conformation and, in turn, alters the conformation
of the G protein.
3. Altering the conformation of the alpha subunit
allows it to exchange GDP for GTP.
4. The binding of GTP to the alpha subunit causes it
to dissociate from the beta subunit.
5. These are now two independent entities: the alpha
subunit and the beta-gamma (bg) subunit.
6. GTP binding and dissociation of the bg subunit
causes the alpha subunit to adopt a new
conformation. The alpha subunit can now
bind to intracellular signaling proteins.
7. The bg subunit does not change
conformation, but its dissociation from the
alpha exposes this face of the beta subunit.
It now can also interact with signaling
molecules.
Inactivation of G proteins
1. The active alpha subunit binds to its target protein
and activates it. Common targets of alpha subunits
are enzymes and ion channels.
2. The internal GTPase activity of the alpha subunit
cleaves the GTP to GDP.
3. This conversion to GDP inactivates the alpha
subunit, causing it to dissociate from its target
protein.
4. The inactive alpha subunit can then associate again
with the bg subunit, reforming the inactive G protein.

**G proteins are usually active for very short periods of

time (seconds).
**Alpha subunits have intrinsically weak GTPase activity,
and on their own would take minutes to hydrolyze
their GTP.
**GTPase activating proteins (GAPs), when bound to
the alpha subunit, enhance the GTPase activity
tremendously. GAPs are critical negative regulators
of G proteins.
**Regulator of G protein signaling (RGS) proteins are
a large family of GAPs that are thought to be
required for turning off G protein cascades in ALL
eukaryotes.
**Each RGS protein has preferred alphas that they
regulate.
Some G proteins signal via cyclic AMP (cAMP)
**Stimulatory G proteins (Gs) activate an enzyme called adenylyl cyclase, which produces cAMP
from ATP. Adenylyl cyclase is a transmembrane protein with its enzymatic region in the cytoplasm.
**This image depicts a neuron that has been stimulated with serotonin, which is activating a GPCR
that is coupled to Gs. The image shows the monitoring of cAMP levels with a fluorescent method.
Red indicates an increase of cAMP by 20-fold within 20 seconds.
**There are inhibitory G proteins (Gi) that inhibit adenylyl cyclase, thereby stopping the
production of cAMP.
**cAMP phosphodiesterases hydrolyze cAMP to AMP, thereby counter-acting the production of
cAMP. Often cAMP levels rapidly decline after production due to cAMP phosphodiesterases.
Production of cAMP

1. Adenylyl cyclase produces cAMP by removing

two phosphate groups from ATP.
2. The phosphates are removed as pyrophosphate
(P-P).
3. Along with the removal of pyrophosphate the
molecule is cyclized.
4. cAMP phosphodiesterase then hydrolyzes
cAMP to AMP.

Cholera toxin: inactivates the GTPase activity of

the Gs alpha subunit, thereby keeping it active. This
causes oversecretion of chloride ions and water into
the gut (severe diahrrhea).

Pertussis toxin: this toxin inactivates the alpha

subunit of Gi. This blocks its ability to
negatively regulate its targets (whooping cough).
Most effects of cAMP are mediated by protein kinase A (PKA)
1. PKA is activated by cAMP and mediates the majority of cAMP effects.
2. There are many different substrates of PKA in different cells, which may explain why rises
in cAMP in different cell types result in very different responses.
3. The inactive form of PKA is a heterotetramer of two catalytic (kinase) subunits and two
regulatory subunits.
4. The binding of cAMP to the regulatory subunits causes a conformational change that
releases the catalytic subunits.
5. The release of the catalytic subunits activates them, allowing them to phosphorylate their
substrates on serine and threonine residues.
Rapid and Slow responses to PKA
activation
**Some of the effects of PKA activation are rapid.
Example: the stimulation of glycogen breakdown to
glucose in muscle cells. This occurs by the direct
phosphorylation of proteins involved in glycogen
metabolism. This provides glucose for energy
production in muscle cells within seconds.
**Some of the effects of PKA are slower. Example: the
activation of gene expression, such as the
somatostatin hormone.
• Activated PKA can translocate into the nucleus.
• There it phosphorylates the transcription factor CREB
(cAMP response element binding protein).
3. When CREB is phosphorylated it binds to the cAMP
response element (CRE).
4. CREB-binding protein (CBP) binds to phosphorylated
CREB and activates transcription of genes that contain
CRE sequences, such as the somatostatin gene.
5. Many cAMP-induced genes contain CRE sequences
and are regulated by CREB and CBP.
Production of inositol phospholipids
**Phosphatylinositol (PI) 4-phosphate and PI 4,5 bisphosphate are produced by the sequential
actions of PI kinase and PIP kinase, respectively.
**PIs exist in the inner leaflet of the plasma membrane.
**PI 4,5 bisphosphate is especially important because its breakdown produces two different
second messengers.
**PI 4,5 bisphosphate is the least abundant of the PIs, and accounts for only 1% of total
phospholipids.
Phospholipase C-b is critical for
GPCR signaling

1. The G protein q (Gq) alpha subunit activates

the enzyme phospholipase C-b (PLC-b) in a
manner similar to how Gs activates adenylyl
cyclase.
2. Activated PLC-b cleaves PI 4,5 bisphosphate
to produce diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (IP3).
3. Importantly, both DAG and IP3 are second
messengers that activate distinct intracellular
signaling molecules.
4. IP3 is a small, water-soluble molecule that
readily diffuses through the cytosol.
5. DAG remains embedded in the plasma
membrane, but like other PM lipids, can
diffuse laterally through the membrane.
The targets of IP3 and DAG
IP3: When IP3 diffuses to the membrane of the ER it binds to IP3-gated calcium release
channels (IP3Rs) in the membrane, triggering their opening.
**IP3Rs release calcium from the ER into the cytosol, rapidly increasing the concentration of
calcium in the cytosol. Ca2+ is perhaps the most common second messenger in cells.
**Calcium levels are quickly reduced by channels that pump it out of the cell, and by the
inactivation of IP3 by dephosphorylation and other means.
DAG: DAG has two signaling functions. First, it can be further cleaved to arachidonic acid,
which can initiate a complex cascade of lipid messengers. Second, DAG can activate a
serine/threonine kinase called protein kinase C (PKC).
**PKC requires both Ca2+ and DAG, along with membrane phospholipids, to be activated.
**PKC has numerous protein substrates that are unique from PKA.
Calcium is a ubiquitous second messenger
1. Ca2+ is a second messenger derived from the activation of many types of cell surface
proteins, not just GPCRs.
2. Calcium levels in the cytosol is very low (10-7M) and very high extracellularly (10-3M).
Calcium levels are also high in the ER.
3. Therefore, modest amounts of calcium entering the cytosol can change the concentration
very rapidly by 10-20 fold.
4. This is an image of an egg cell that is being fertilized by a sperm. This process triggers a
wave of calcium release from the ER from the site of contact with the sperm. This wave of
calcium triggers the beginning of embryonic development.
Calmodulin
1. Calmodulin is a Ca2+ binding protein that has 4 high affinity binding sites for Ca2+.
2. Calmodulin is extremely abundant in cells and accounts for as much as 1% of total protein.
3. Binding of calcium causes a conformational change in calmodulin.
4. At least two or more Ca2+ must bind before calmodulin changes conformations, making it
behave like a switch to increasing concentrations of calcium.
5. Calmodulin has no enzymatic function, and instead binds to target proteins and alters their
confirmation (as well as its own).
6. One of the most important group of calmodulin targets is the Ca2+/calmodulin-dependent
protein kinases (CaM-kinases).
CaM-kinase II
1. CaM-kinase II is composed of a large
complex of about 12 subunits of CaM-
kinase II. For simplicity, only one is
shown here.
2. Upon Ca2+/calmodulin binding, CaMKII
changes conformation and is activated.
3. Upon activation, CaMKII
autophosphorylates itself on an
autoinhibitory domain. This
phosphorylation event sustains CaMKII
activity without Ca2+/calmodulin being
present in two ways. First, it locks
Ca2+/calmodulin binding to it such that it
will not dissociate without the prolonged
return to normal calcium levels. Second,
it converts the enzyme to a calcium
independent form.
4. After this occurs, CaMKII can only be
inactivated if all of the subunits are
dephosphorylated by phosphatases
(overriding the CaMKII kinase activity).
CaMKII is a “frequency decoder” for calcium waves
1. During low-frequency calcium spikes (or oscillations), CaMKII does not remain active long
enough before the next calcium spike, and there is no overall difference in subsequent
CaMKII activations.
2. During high frequency calcium waves, the amount of CaMKII activity ratchets up upon
each successive calcium spike because it never fully inactivated. This “ratcheting” of
CaMKII activity will continue until all 12 subunits are fully activated. It will remain active for
a long period of time, even after the calcium spikes stop.
**This is a type of “cellular memory” because CaMKII can “remember” prior calcium spikes.
**CaMKII is critical for spatial memory in the brain (where CaMKII is as much as 2% of brain
protein!!). It is this quality of calcium frequency decoding that enables it to encode
memories.
GPCR desensitization
**Cells desensitize, or adapt, when exposed to high levels of ligand for a long period of time.
There are 3 mechanisms of desensitization at the level of the GPCR:
1. Receptor inactivation: The GPCR becomes modified such that it can no longer interact
with its G protein.
2. Receptor sequestration: The GPCR can be internalized and transported to an interior
compartment of the cell such that it no longer is exposed to ligand.
3. Receptor downregulation: The receptor can be degraded by lysosomes after it is
internalized.
G-protein-linked receptor kinases (GRKs): phosphorylate GPCRs upon their activation on
multiple serines and threonines.
This phosphorylation leads to binding of arrestin to the active GPCR. Arrestin triggers
desensitization by 1). inhibiting the binding of the G protein to the GPCR and 2). by
acting as an adaptor protein for the internalization of the receptor. Whether the receptor
is sequestered or degraded depends upon may factors.