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GPCR |
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Available protein structures: |
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The seven-transmembrane α-helix structure of a G protein-coupled receptor
G protein-coupled receptors (
GPCRs), also known as
seven-(pass)-transmembrane domain receptors,
7TM receptors,
heptahelical receptors,
serpentine receptor, and
G protein–linked receptors (
GPLR), constitute a large
protein family of
receptors that detect
molecules outside the
cell and activate internal
signal transduction pathways and, ultimately, cellular responses. Coupling with
G proteins, they are called seven-transmembrane receptors because they pass through the
cell membrane seven times.
G protein-coupled receptors are found only in
eukaryotes, including
yeast,
choanoflagellates, and animals. The
ligands that bind and activate these receptors include light-sensitive compounds,
odors,
pheromones,
hormones, and
neurotransmitters, and vary in size from small molecules to
peptides to large
proteins.
G protein-coupled receptors are involved in many diseases, and are also
the target of approximately 34% of all modern medicinal drugs.
There are two principal signal transduction pathways involving the G protein-coupled receptors:
When a ligand binds to the GPCR it causes a conformational change in the GPCR, which allows it to act as a
guanine nucleotide exchange factor (GEF). The GPCR can then activate an associated
G protein by exchanging the
GDP bound to the G protein for a
GTP.
The G protein's α subunit, together with the bound GTP, can then
dissociate from the β and γ subunits to further affect intracellular
signaling proteins or target functional proteins directly depending on
the α subunit type (
Gαs,
Gαi/o,
Gαq/11,
Gα12/13).
GPCRs are an important drug target and approximately 34%
of all Food and Drug Administration (FDA) approved drugs target 108
members of this family. The global sales volume for these drugs is
estimated to be 180 billion US dollars as of 2018.
History and significance
The 2012
Nobel Prize in Chemistry was awarded to
Brian Kobilka and
Robert Lefkowitz for their work that was "crucial for understanding how G protein-coupled receptors function". There have been at least
seven other Nobel Prizes awarded for some aspect of G protein–mediated signaling. As of 2012, two of the top ten global best-selling drugs (
Advair Diskus and
Abilify) act by targeting G protein-coupled receptors.
Classification
Classification
Scheme of GPCRs. Class A (Rhodopsin-like), Class B (Secretin-like),
Class C (Glutamate Receptor-like), Others (Adhesion (33), Frizzled (11),
Taste type-2 (25), unclassified (23)).
The exact size of the GPCR superfamily is unknown, but nearly 800 different
human genes (or ~ 4% of the entire
protein-coding genome) have been predicted to code for them from genome
sequence analysis.
Although numerous classification schemes have been proposed, the
superfamily was classically divided into three main classes (A, B, and
C) with no detectable shared
sequence homology between classes.
The largest class by far is class A, which accounts for nearly
85% of the GPCR genes. Of class A GPCRs, over half of these are
predicted to encode
olfactory receptors, while the remaining receptors are
liganded by known
endogenous compounds or are classified as
orphan receptors. Despite the lack of sequence homology between classes, all GPCRs have a common
structure and mechanism of
signal transduction. The very large rhodopsin A group has been further subdivided into 19 subgroups (
A1-A19).
More recently, an alternative classification system called GRAFS (
Glutamate,
Rhodopsin,
Adhesion,
Frizzled/
Taste2,
Secretin) has been proposed.
According to the classical A-F system, GPCRs can be grouped into 6
classes based on sequence homology and functional similarity:
An early study based on available DNA sequence suggested that the human genome encodes roughly 750 G protein-coupled receptors,
about 350 of which detect hormones, growth factors, and other
endogenous ligands. Approximately 150 of the GPCRs found in the human
genome have unknown functions.
Some web-servers and bioinformatics prediction methods have been used for predicting the classification of GPCRs according to their amino acid sequence alone, by means of the
pseudo amino acid composition approach.
Physiological roles
GPCRs are involved in a wide variety of physiological processes. Some examples of their physiological roles include:
- The visual sense: The opsins, gradually evolved from early GPCRs over 650 million years ago, use a photoisomerization reaction to translate electromagnetic radiation into cellular signals. Rhodopsin, for example, uses the conversion of 11-cis-retinal to all-trans-retinal for this purpose.
- The gustatory sense (taste): GPCRs in taste cells mediate release of gustducin in response to bitter-, umami- and sweet-tasting substances.
- The sense of smell: Receptors of the olfactory epithelium bind odorants (olfactory receptors) and pheromones (vomeronasal receptors)
- Behavioral and mood regulation: Receptors in the mammalian brain bind several different neurotransmitters, including serotonin, dopamine, GABA, and glutamate
- Regulation of immune system activity and inflammation: Chemokine receptors bind ligands that mediate intercellular communication between cells of the immune system; receptors such as histamine receptors bind inflammatory mediators and engage target cell types in the inflammatory response.
GPCRs are also involved in immune-modulation and directly involved in
suppression of TLR-induced immune responses from T cells.
- Autonomic nervous system transmission: Both the sympathetic and parasympathetic
nervous systems are regulated by GPCR pathways, responsible for control
of many automatic functions of the body such as blood pressure, heart
rate, and digestive processes
- Cell density sensing: A novel GPCR role in regulating cell density sensing.
- Homeostasis modulation (e.g., water balance).
- Involved in growth and metastasis of some types of tumors.
- Used in the endocrine system for peptide and amino-acid derivative
hormones that bind to GCPRs on the cell membrane of a target cell. This
activates cAMP, which in turn activates several kinases, allowing for a
cellular response, such as transcription.
Receptor structure
GPCRs are
integral membrane proteins that possess seven membrane-spanning domains or
transmembrane helices.
[25][26] The extracellular parts of the receptor can be
glycosylated. These extracellular loops also contain two highly conserved
cysteine residues that form
disulfide bonds to stabilize the receptor structure. Some seven-transmembrane helix proteins (
channelrhodopsin) that resemble GPCRs may contain ion channels, within their protein.
In 2000, the first crystal structure of a mammalian GPCR, that of bovine
rhodopsin (
1F88), was solved. In 2007, the first structure of a human GPCR was solved. This human
β2-adrenergic receptor
GPCR structure proved highly similar to the bovine rhodopsin. The
structures of activated or agonist-bound GPCRs have also been
determined.
These structures indicate how ligand binding at the extracellular side
of a receptor leads to conformational changes in the cytoplasmic side of
the receptor. The biggest change is an outward movement of the
cytoplasmic part of the 5th and 6th transmembrane helix (TM5 and TM6).
The structure of activated beta-2 adrenergic receptor in complex with G
s confirmed that the Gα binds to a cavity created by this movement.
GPCR are evolutionarily related to some other proteins with 7
transmembrane domains, such as
microbial rhodopsins and adiponectin receptors 1 and 2 (
ADIPOR1 and
ADIPOR2). However, these 7TMH receptors and channels do not associate with
G proteins. In addition, ADIPOR1 and ADIPOR2 are oriented oppositely to GPCRs in the membrane (i.e. GPCRs usually have an extracellular
N-terminus, cytoplasmic
C-terminus, whereas ADIPORs are inverted).
Structure-function relationships
Two-dimensional
schematic of a generic GPCR set in a Lipid
Raft. Click the image for
higher resolution to see details
regarding the locations of important
structures.
In terms of structure, GPCRs are characterized by an extracellular
N-terminus, followed by seven
transmembrane (7-TM)
α-helices
(TM-1 to TM-7) connected by three intracellular (IL-1 to IL-3) and
three extracellular loops (EL-1 to EL-3), and finally an intracellular
C-terminus. The GPCR arranges itself into a
tertiary structure resembling a barrel, with the seven transmembrane helices forming a cavity within the plasma membrane that serves a
ligand-binding
domain that is often covered by EL-2. Ligands may also bind elsewhere,
however, as is the case for bulkier ligands (e.g.,
proteins or large
peptides), which instead interact with the extracellular loops, or, as illustrated by the class C
metabotropic glutamate receptors
(mGluRs), the N-terminal tail. The class C GPCRs are distinguished by
their large N-terminal tail, which also contains a ligand-binding
domain. Upon glutamate-binding to an mGluR, the N-terminal tail
undergoes a conformational change that leads to its interaction with the
residues of the extracellular loops and TM domains. The eventual effect
of all three types of
agonist-induced
activation is a change in the relative orientations of the TM helices
(likened to a twisting motion) leading to a wider intracellular surface
and "revelation" of residues of the intracellular helices and TM domains
crucial to signal transduction function (i.e., G-protein coupling).
Inverse agonists and
antagonists may also bind to a number of different sites, but the eventual effect must be prevention of this TM helix reorientation.
The structure of the N- and C-terminal tails of GPCRs may also
serve important functions beyond ligand-binding. For example, The
C-terminus of M
3 muscarinic receptors is sufficient, and the
six-amino-acid polybasic (KKKRRK) domain in the C-terminus is necessary
for its preassembly with G
q proteins. In particular, the C-terminus often contains
serine (Ser) or
threonine (Thr) residues that, when
phosphorylated, increase the
affinity of the intracellular surface for the binding of scaffolding proteins called β-
arrestins (β-arr). Once bound, β-arrestins both
sterically
prevent G-protein coupling and may recruit other proteins, leading to
the creation of signaling complexes involved in extracellular-signal
regulated kinase (
ERK) pathway activation or receptor
endocytosis
(internalization). As the phosphorylation of these Ser and Thr residues
often occurs as a result of GPCR activation, the β-arr-mediated
G-protein-decoupling and internalization of GPCRs are important
mechanisms of
desensitization. In addition, internalized "mega-complexes" consisting of a single GPCR, β-arr(in the tail conformation), and heterotrimeric G protein exist and may account for protein signaling from endosomes.
A final common structural theme among GPCRs is
palmitoylation of one or more sites of the C-terminal tail or the intracellular loops. Palmitoylation is the covalent modification of
cysteine (Cys) residues via addition of hydrophobic
acyl groups, and has the effect of targeting the receptor to
cholesterol- and
sphingolipid-rich microdomains of the plasma membrane called
lipid rafts. As many of the downstream transducer and effector molecules of GPCRs (including those involved in
negative feedback pathways) are also targeted to lipid rafts, this has the effect of facilitating rapid receptor signaling.
GPCRs respond to extracellular signals mediated by a huge diversity of agonists, ranging from proteins to
biogenic amines to
protons, but all transduce this signal via a mechanism of G-protein coupling. This is made possible by a
guanine-nucleotide exchange factor (
GEF) domain primarily formed by a combination of IL-2 and IL-3 along with adjacent residues of the associated TM helices.
Mechanism
Cartoon
depicting the basic concept of GPCR conformational activation. Ligand
binding disrupts an ionic lock between the E/DRY motif of TM-3 and
acidic residues of TM-6. As a result, the GPCR reorganizes to allow
activation of G-alpha proteins. The side perspective is a view from
above and to the side of the GPCR as it is set in the plasma membrane
(the membrane lipids have been omitted for clarity). The intracellular
perspective shows the view looking up at the plasma membrane from inside
the cell.
The G protein-coupled receptor is activated by an external signal in
the form of a ligand or other signal mediator. This creates a
conformational change in the receptor, causing activation of a
G protein.
Further effect depends on the type of G protein. G proteins are
subsequently inactivated by GTPase activating proteins, known as
RGS proteins.
Ligand binding
GPCRs include one or more receptors for the following ligands:
sensory signal mediators (e.g., light and
olfactory stimulatory molecules);
adenosine,
bombesin,
bradykinin,
endothelin, γ-aminobutyric acid (
GABA), hepatocyte growth factor (
HGF),
melanocortins,
neuropeptide Y,
opioid peptides,
opsins,
somatostatin,
GH,
tachykinins, members of the
vasoactive intestinal peptide family, and
vasopressin;
biogenic amines (e.g.,
dopamine,
epinephrine,
norepinephrine,
histamine,
serotonin, and
melatonin);
glutamate (
metabotropic effect);
glucagon;
acetylcholine (
muscarinic effect);
chemokines;
lipid mediators of
inflammation (e.g.,
prostaglandins,
prostanoids,
platelet-activating factor, and
leukotrienes);
peptide hormones (e.g.,
calcitonin, C5a
anaphylatoxin, follicle-stimulating hormone [
FSH], gonadotropin-releasing hormone [
GnRH],
neurokinin, thyrotropin-releasing hormone [
TRH], and
oxytocin);
and
endocannabinoids.
GPCRs that act as receptors for stimuli that have not yet been identified are known as
orphan receptors.
However, in other types of receptors that have been studied, wherein ligands bind externally to the membrane, the
ligands of GPCRs typically bind within the transmembrane domain. However,
protease-activated receptors are activated by cleavage of part of their extracellular domain.
Conformational change
Crystal structure of activated beta-2 adrenergic receptor in complex with G
s(
PDB entry
3SN6).
The receptor is colored red, Gα green, Gβ cyan, and Gγ yellow. The
C-terminus of Gα is located in a cavity created by an outward movement
of the cytoplasmic parts of TM5 and 6.
The
transduction of the signal
through the membrane by the receptor is not completely understood. It
is known that in the inactive state, the GPCR is bound to a
heterotrimeric G protein complex. Binding of an agonist to the GPCR results in a
conformational change in the receptor that is transmitted to the bound G
α subunit of the heterotrimeric G protein via
protein domain dynamics. The activated G
α subunit exchanges
GTP in place of
GDP which in turn triggers the dissociation of G
α subunit from the G
βγ dimer and from the receptor. The dissociated G
α and G
βγ
subunits interact with other intracellular proteins to continue the
signal transduction cascade while the freed GPCR is able to rebind to
another heterotrimeric G protein to form a new complex that is ready to
initiate another round of signal transduction.
It is believed that a receptor molecule exists in a conformational
equilibrium between active and inactive biophysical states.
The binding of ligands to the receptor may shift the equilibrium toward
the active receptor states. Three types of ligands exist: Agonists are
ligands that shift the equilibrium in favour of active states;
inverse agonists
are ligands that shift the equilibrium in favour of inactive states;
and neutral antagonists are ligands that do not affect the equilibrium.
It is not yet known how exactly the active and inactive states differ
from each other.
G-protein activation/deactivation cycle
Cartoon depicting the Heterotrimeric G-protein activation/
deactivation cycle in the context of GPCR signaling
When the receptor is inactive, the
GEF domain may be bound to an also inactive α-subunit of a
heterotrimeric G-protein. These "G-proteins" are a
trimer of α, β, and γ subunits (known as Gα, Gβ, and Gγ, respectively) that is rendered inactive when reversibly bound to
Guanosine diphosphate (GDP) (or, alternatively, no guanine nucleotide) but active when bound to
guanosine triphosphate (GTP). Upon receptor activation, the GEF domain, in turn,
allosterically
activates the G-protein by facilitating the exchange of a molecule of
GDP for GTP at the G-protein's α-subunit. The cell maintains a 10:1
ratio of cytosolic GTP:GDP so exchange for GTP is ensured. At this
point, the subunits of the G-protein dissociate from the receptor, as
well as each other, to yield a Gα-GTP
monomer and a tightly interacting
Gβγ dimer, which are now free to modulate the activity of other intracellular proteins. The extent to which they may
diffuse, however, is limited due to the
palmitoylation of Gα and the presence of an
isoprenoid moiety that has been
covalently added to the C-termini of Gγ.
Because Gα also has slow
GTP→GDP hydrolysis
capability, the inactive form of the α-subunit (Gα-GDP) is eventually
regenerated, thus allowing reassociation with a Gβγ dimer to form the
"resting" G-protein, which can again bind to a GPCR and await
activation. The rate of GTP hydrolysis is often accelerated due to the
actions of another family of allosteric modulating proteins called
Regulators of G-protein Signaling, or RGS proteins, which are a type of
GTPase-Activating Protein, or GAP. In fact, many of the primary
effector proteins (e.g.,
adenylate cyclases)
that become activated/inactivated upon interaction with Gα-GTP also
have GAP activity. Thus, even at this early stage in the process,
GPCR-initiated signaling has the capacity for self-termination.
Crosstalk
Proposed downstream interactions between
integrin signaling and GPCRs. Integrins are shown elevating Ca
2+ and phosphorylating FAK, which is weakening GPCR signaling.
GPCRs downstream signals have been shown to possibly interact with
integrin signals, such as
FAK. Integrin signaling will phosphorylate FAK, which can then decrease GPCR Gαs activity.
Signaling
G-protein-coupled receptor mechanism
If a receptor in an active state encounters a
G protein, it may activate it. Some evidence suggests that receptors and G proteins are actually pre-coupled. For example, binding of G proteins to receptors affects the receptor's affinity for ligands. Activated G proteins are bound to
GTP.
Further signal transduction depends on the type of G protein. The enzyme
adenylate cyclase is an example of a cellular protein that can be regulated by a G protein, in this case the G protein
Gs. Adenylate cyclase activity is activated when it binds to a subunit of
the activated G protein. Activation of adenylate cyclase ends when the G
protein returns to the
GDP-bound state.
Adenylate cyclases (of which 9 membrane-bound and one cytosolic
forms are known in humans) may also be activated or inhibited in other
ways (e.g., Ca2+/
Calmodulin binding), which can modify the activity of these enzymes in an additive or synergistic fashion along with the G proteins.
The signaling pathways activated through a GPCR are limited by the
primary sequence and
tertiary structure of the GPCR itself but ultimately determined by the particular
conformation stabilized by a particular
ligand, as well as the availability of
transducer molecules. Currently, GPCRs are considered to utilize two primary types of transducers:
G-proteins and
β-arrestins. Because β-arr's have high
affinity only to the
phosphorylated
form of most GPCRs (see above or below), the majority of signaling is
ultimately dependent upon G-protein activation. However, the possibility
for interaction does allow for G-protein-independent signaling to
occur.
G-protein-dependent signaling
There are three main G-protein-mediated signaling pathways, mediated by four
sub-classes of G-proteins distinguished from each other by
sequence homology (
Gαs,
Gαi/o,
Gαq/11, and
Gα12/13). Each sub-class of G-protein consists of multiple proteins, each the product of multiple
genes or
splice variations
that may imbue them with differences ranging from subtle to distinct
with regard to signaling properties, but in general they appear
reasonably grouped into four classes. Because the signal transducing
properties of the various possible
βγ combinations do not appear to radically differ from one another, these classes are defined according to the isoform of their α-subunit.
While most GPCRs are capable of activating more than one
Gα-subtype, they also show a preference for one subtype over another.
When the subtype activated depends on the ligand that is bound to the
GPCR, this is called
functional selectivity (also known as agonist-directed trafficking, or conformation-specific agonism). However, the binding of any single particular
agonist
may also initiate activation of multiple different G-proteins, as it
may be capable of stabilizing more than one conformation of the GPCR's
GEF domain, even over the course of a single interaction. In addition, a conformation that preferably activates one
isoform of Gα may activate another if the preferred is less available. Furthermore,
feedback pathways may result in
receptor modifications
(e.g., phosphorylation) that alter the G-protein preference. Regardless
of these various nuances, the GPCR's preferred coupling partner is
usually defined according to the G-protein most obviously activated by
the
endogenous ligand under most
physiological or
experimental conditions.
Gα signaling
- The effector of both the Gαs and Gαi/o pathways is the cyclic-adenosine monophosphate (cAMP)-generating enzyme adenylate cyclase, or AC. While there are ten different AC gene products in mammals, each with subtle differences in tissue distribution or function, all catalyze the conversion of cytosolic adenosine triphosphate (ATP) to cAMP, and all are directly stimulated by G-proteins of the Gαs class. In contrast, however, interaction with Gα subunits of the Gαi/o type inhibits AC from generating cAMP. Thus, a GPCR coupled to Gαs counteracts the actions of a GPCR coupled to Gαi/o, and vice versa. The level of cytosolic cAMP may then determine the activity of various ion channels as well as members of the ser/thr-specific protein kinase A (PKA) family. Thus cAMP is considered a second messenger and PKA a secondary effector.
- The effector of the Gαq/11 pathway is phospholipase C-β (PLCβ), which catalyzes the cleavage of membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 acts on IP3 receptors found in the membrane of the endoplasmic reticulum (ER) to elicit Ca2+ release from the ER, while DAG diffuses along the plasma membrane where it may activate any membrane localized forms of a second ser/thr kinase called protein kinase C (PKC). Since many isoforms of PKC are also activated by increases in intracellular Ca2+, both these pathways can also converge on each other to signal through the same secondary effector. Elevated intracellular Ca2+ also binds and allosterically activates proteins called calmodulins, which in turn go on to bind and allosterically activate enzymes such as Ca2+/calmodulin-dependent kinases (CAMKs).
- The effectors of the Gα12/13 pathway are three RhoGEFs (p115-RhoGEF, PDZ-RhoGEF, and LARG), which, when bound to Gα12/13 allosterically activate the cytosolic small GTPase, Rho. Once bound to GTP, Rho can then go on to activate various proteins responsible for cytoskeleton regulation such as Rho-kinase (ROCK). Most GPCRs that couple to Gα12/13 also couple to other sub-classes, often Gαq/11.
Gβγ signaling
The above descriptions ignore the effects of
Gβγ–signalling, which can also be important, in particular in the case of activated G
αi/o-coupled GPCRs. The primary effectors of Gβγ are various ion channels, such as
G-protein-regulated inwardly rectifying K+ channels (GIRKs),
P/
Q- and
N-type voltage-gated Ca2+ channels, as well as some isoforms of AC and PLC, along with some
phosphoinositide-3-kinase (PI3K) isoforms.
G-protein-independent signaling
Although
they are classically thought of working only together, GPCRs may signal
through G-protein-independent mechanisms, and heterotrimeric G-proteins
may play functional roles independent of GPCRs. GPCRs may signal
independently through many proteins already mentioned for their roles in
G-protein-dependent signaling such as
β-arrs,
GRKs, and
Srcs. In addition, further scaffolding proteins involved in
subcellular localization of GPCRs (e.g.,
PDZ-domain-containing proteins) may also act as signal transducers. Most often the effector is a member of the
MAPK family.
Examples
In the late 1990s, evidence began accumulating to suggest that some GPCRs are able to signal without G proteins. The
ERK2
mitogen-activated protein kinase, a key signal transduction mediator
downstream of receptor activation in many pathways, has been shown to be
activated in response to cAMP-mediated receptor activation in the
slime mold D. discoideum despite the absence of the associated G protein α- and β-subunits.
In mammalian cells, the much-studied β
2-adrenoceptor
has been demonstrated to activate the ERK2 pathway after
arrestin-mediated uncoupling of G-protein-mediated signaling. Therefore,
it seems likely that some mechanisms previously believed related purely
to receptor desensitisation are actually examples of receptors
switching their signaling pathway, rather than simply being switched
off.
In kidney cells, the
bradykinin receptor B2 has been shown to interact directly with a protein tyrosine phosphatase. The presence of a tyrosine-phosphorylated
ITIM
(immunoreceptor tyrosine-based inhibitory motif) sequence in the B2
receptor is necessary to mediate this interaction and subsequently the
antiproliferative effect of bradykinin.
GPCR-independent signaling by heterotrimeric G-proteins
Although
it is a relatively immature area of research, it appears that
heterotrimeric G-proteins may also take part in non-GPCR signaling.
There is evidence for roles as signal transducers in nearly all other
types of receptor-mediated signaling, including
integrins,
receptor tyrosine kinases (RTKs),
cytokine receptors (
JAK/STATs), as well as modulation of various other "accessory" proteins such as
GEFs,
guanine-nucleotide dissociation inhibitors (GDIs) and
protein phosphatases.
There may even be specific proteins of these classes whose primary
function is as part of GPCR-independent pathways, termed activators of
G-protein signalling (AGS). Both the ubiquity of these interactions and
the importance of Gα vs. Gβγ subunits to these processes are still
unclear.
Details of cAMP and PIP2 pathways
Activation effects of cAMP on protein kinase A
The effect of Rs and Gs in cAMP signal pathway
The effect of Ri and Gi in cAMP signal pathway
There are two principal signal transduction pathways involving the
G protein-linked receptors: the
cAMP signal pathway and the
phosphatidylinositol signal pathway.
cAMP signal pathway
The cAMP signal transduction contains 5 main characters: stimulative
hormone receptor (Rs) or inhibitory
hormone receptor (Ri); stimulative regulative G-protein (Gs) or inhibitory regulative G-protein (Gi);
adenylyl cyclase;
protein kinase A (PKA); and cAMP
phosphodiesterase.
Stimulative hormone receptor (Rs) is a receptor that can bind
with stimulative signal molecules, while inhibitory hormone receptor
(Ri) is a receptor that can bind with inhibitory signal molecules.
Stimulative regulative G-protein is a G-protein linked to
stimulative hormone receptor (Rs), and its α subunit upon activation
could stimulate the activity of an enzyme or other intracellular
metabolism. On the contrary, inhibitory regulative G-protein is linked
to an inhibitory hormone receptor, and its α subunit upon activation
could inhibit the activity of an enzyme or other intracellular
metabolism.
Adenylyl cyclase is a 12-transmembrane glycoprotein that catalyzes ATP to form cAMP with the help of cofactor Mg
2+ or Mn
2+. The cAMP produced is a second messenger in cellular metabolism and is an allosteric activator of protein kinase A.
Protein kinase A is an important enzyme in cell metabolism due to
its ability to regulate cell metabolism by phosphorylating specific
committed enzymes in the metabolic pathway. It can also regulate
specific gene expression, cellular secretion, and membrane permeability.
The protein enzyme contains two catalytic subunits and two regulatory
subunits. When there is no cAMP,the complex is inactive. When cAMP binds
to the regulatory subunits, their conformation is altered, causing the
dissociation of the regulatory subunits, which activates protein kinase A
and allows further biological effects.
These signals then can be terminated by cAMP phosphodiesterase,
which is an enzyme that degrades cAMP to 5'-AMP and inactivates protein
kinase A.
Phosphatidylinositol signal pathway
In the
phosphatidylinositol signal pathway, the extracellular signal molecule binds with the G-protein receptor (G
q) on the cell surface and activates
phospholipase C, which is located on the
plasma membrane. The
lipase hydrolyzes
phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers:
inositol 1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG). IP3 binds with the
IP3 receptor in the membrane of the smooth endoplasmic reticulum and mitochondria to open Ca
2+ channels. DAG helps activate
protein kinase C (PKC), which phosphorylates many other proteins, changing their catalytic activities, leading to cellular responses.
The effects of Ca
2+ are also remarkable: it cooperates with DAG in activating PKC and can activate the
CaM kinase pathway, in which calcium-modulated protein
calmodulin (CaM) binds Ca
2+,
undergoes a change in conformation, and activates CaM kinase II, which
has unique ability to increase its binding affinity to CaM by
autophosphorylation, making CaM unavailable for the activation of other
enzymes. The kinase then phosphorylates target enzymes, regulating their
activities. The two signal pathways are connected together by Ca
2+-CaM, which is also a regulatory subunit of adenylyl cyclase and phosphodiesterase in the cAMP signal pathway.
Receptor regulation
GPCRs
become desensitized when exposed to their ligand for a long period of
time. There are two recognized forms of desensitization: 1)
homologous desensitization, in which the activated GPCR is downregulated; and 2)
heterologous desensitization, wherein the activated GPCR causes downregulation of a different GPCR. The key reaction of this downregulation is the
phosphorylation of the intracellular (or
cytoplasmic) receptor domain by
protein kinases.
Phosphorylation by cAMP-dependent protein kinases
Cyclic AMP-dependent protein kinases (
protein kinase A) are activated by the signal chain coming from the G protein (that was activated by the receptor) via
adenylate cyclase and
cyclic AMP (cAMP). In a
feedback mechanism,
these activated kinases phosphorylate the receptor. The longer the
receptor remains active the more kinases are activated and the more
receptors are phosphorylated. In
β2-adrenoceptors, this phosphorylation results in the switching of the coupling from the G
s class of G-protein to the
Gi class. cAMP-dependent PKA mediated phosphorylation can cause heterologous desensitisation in receptors other than those activated.
Phosphorylation by GRKs
The
G protein-coupled receptor kinases (GRKs) are protein kinases that phosphorylate only active GPCRs.
G-protein-coupled receptor kinases (GRKs) are key modulators of
G-protein-coupled receptor (GPCR) signaling. They constitute a family of
seven mammalian serine-threonine protein kinases that phosphorylate
agonist-bound receptor. GRKs-mediated receptor phosphorylation rapidly
initiates profound impairment of receptor signaling and desensitization.
Activity of GRKs and subcellular targeting is tightly regulated by
interaction with receptor domains, G protein subunits, lipids, anchoring
proteins and calcium-sensitive proteins.
Phosphorylation of the receptor can have two consequences:
- Translocation: The receptor is, along with the part of
the membrane it is embedded in, brought to the inside of the cell, where
it is dephosphorylated within the acidic vesicular environment
and then brought back. This mechanism is used to regulate long-term
exposure, for example, to a hormone, by allowing resensitisation to
follow desensitisation. Alternatively, the receptor may undergo
lysozomal degradation, or remain internalised, where it is thought to
participate in the initiation of signalling events, the nature of which
depending on the internalised vesicle's subcellular localisation.
- Arrestin linking: The phosphorylated receptor can be linked to arrestin
molecules that prevent it from binding (and activating) G proteins, in
effect switching it off for a short period of time. This mechanism is
used, for example, with rhodopsin in retina
cells to compensate for exposure to bright light. In many cases,
arrestin's binding to the receptor is a prerequisite for translocation.
For example, beta-arrestin bound to β2-adrenoreceptors acts
as an adaptor for binding with clathrin, and with the beta-subunit of
AP2 (clathrin adaptor molecules); thus, the arrestin here acts as a
scaffold assembling the components needed for clathrin-mediated
endocytosis of β2-adrenoreceptors.
Mechanisms of GPCR signal termination
As mentioned above, G-proteins may terminate their own activation due to their intrinsic
GTP→GDP hydrolysis capability. However, this reaction proceeds at a slow
rate
(≈.02 times/sec) and, thus, it would take around 50 seconds for any
single G-protein to deactivate if other factors did not come into play.
Indeed, there are around 30
isoforms of
RGS proteins that, when bound to Gα through their
GAP domain,
accelerate the hydrolysis rate to ≈30 times/sec. This 1500-fold
increase in rate allows for the cell to respond to external signals with
high speed, as well as spatial
resolution due to limited amount of
second messenger that can be generated and limited distance a G-protein can diffuse in 0.03 seconds. For the most part, the RGS proteins are
promiscuous
in their ability to activate G-proteins, while which RGS is involved in
a given signaling pathway seems more determined by the tissue and GPCR
involved than anything else. In addition, RGS proteins have the
additional function of increasing the rate of GTP-GDP exchange at GPCRs,
(i.e., as a sort of co-GEF) further contributing to the time resolution
of GPCR signaling.
In addition, the GPCR may be
desensitized itself. This can occur as:
- a direct result of ligand occupation, wherein the change in conformation allows recruitment of GPCR-Regulating Kinases (GRKs), which go on to phosphorylate various serine/threonine residues of IL-3 and the C-terminal tail. Upon GRK phosphorylation, the GPCR's affinity for β-arrestin (β-arrestin-1/2 in most tissues) is increased, at which point β-arrestin may bind and act to both sterically hinder G-protein coupling as well as initiate the process of receptor internalization through clathrin-mediated endocytosis. Because only the liganded receptor is desensitized by this mechanism, it is called homologous desensitization
- the affinity for β-arrestin may be increased in a ligand occupation
and GRK-independent manner through phosphorylation of different ser/thr
sites (but also of IL-3 and the C-terminal tail) by PKC and PKA. These
phosphorylations are often sufficient to impair G-protein coupling on
their own as well.
- PKC/PKA may, instead, phosphorylate GRKs, which can also lead to
GPCR phosphorylation and β-arrestin binding in an occupation-independent
manner. These latter two mechanisms allow for desensitization of one
GPCR due to the activities of others, or heterologous desensitization. GRKs may also have GAP domains and so may contribute to inactivation through non-kinase mechanisms as well. A combination of these mechanisms may also occur.
Once β-arrestin is bound to a GPCR, it undergoes a conformational
change allowing it to serve as a scaffolding protein for an adaptor
complex termed
AP-2, which in turn recruits another protein called
clathrin. If enough receptors in the local area recruit clathrin in this manner, they aggregate and the
membrane buds inwardly as a result of interactions between the molecules of clathrin, in a process called
opsonization. Once the pit has been pinched off the
plasma membrane due to the actions of two other proteins called
amphiphysin and
dynamin, it is now an
endocytic vesicle. At this point, the adapter molecules and clathrin have
dissociated, and the receptor is either
trafficked back to the plasma membrane or targeted to
lysosomes for
degradation.
At any point in this process, the β-arrestins may also recruit other proteins—such as the
non-receptor tyrosine kinase (nRTK),
c-SRC—which may activate
ERK1/2, or other
mitogen-activated protein kinase (MAPK) signaling through, for example, phosphorylation of the
small GTPase,
Ras, or recruit the proteins of the
ERK cascade directly (i.e.,
Raf-1,
MEK,
ERK-1/2) at which point signaling is initiated due to their close
proximity to one another. Another target of c-SRC are the dynamin
molecules involved in endocytosis. Dynamins
polymerize
around the neck of an incoming vesicle, and their phosphorylation by
c-SRC provides the energy necessary for the conformational change
allowing the final "pinching off" from the membrane.
GPCR cellular regulation
Receptor
desensitization is mediated through a combination phosphorylation,
β-arr binding, and endocytosis as described above. Downregulation occurs
when endocytosed receptor is embedded in an endosome that is trafficked
to merge with an organelle called a lysosome. Because lysosomal
membranes are rich in proton pumps, their interiors have low pH (≈4.8
vs. the pH≈7.2 cytosol), which acts to denature the GPCRs. In addition,
lysosomes contain many
degradative enzymes,
including proteases, which can function only at such low pH, and so the
peptide bonds joining the residues of the GPCR together may be cleaved.
Whether or not a given receptor is trafficked to a lysosome, detained
in endosomes, or trafficked back to the plasma membrane depends on a
variety of factors, including receptor type and magnitude of the signal.
GPCR regulation is additionally mediated by gene transcription factors.
These factors can increase or decrease gene transcription and thus
increase or decrease the generation of new receptors (up- or
down-regulation) that travel to the cell membrane.
Receptor oligomerization
G-protein-coupled receptor oligomerisation is a widespread phenomenon. One of the best-studied examples is the metabotropic
GABAB receptor. This so-called constitutive receptor is formed by heterodimerization of
GABABR1 and
GABABR2 subunits. Expression of the GABA
BR1 without the GABA
BR2 in heterologous systems leads to retention of the subunit in the
endoplasmic reticulum. Expression of the GABA
BR2
subunit alone, meanwhile, leads to surface expression of the subunit,
although with no functional activity (i.e., the receptor does not bind
agonist and cannot initiate a response following exposure to agonist).
Expression of the two subunits together leads to plasma membrane
expression of functional receptor. It has been shown that GABA
BR2 binding to GABA
BR1 causes masking of a retention signal of functional receptors.
Origin and diversification of the superfamily
Signal
transduction mediated by the superfamily of GPCRs dates back to the
origin of multicellularity. Mammalian-like GPCRs are found in
fungi, and have been classified according to the
GRAFS classification system based on GPCR fingerprints. Identification of the superfamily members across the
eukaryotic domain, and comparison of the family-specific motifs, have shown that the superfamily of GPCRs have a common origin. Characteristic motifs indicate that three of the five GRAFS families,
Rhodopsin,
Adhesion, and
Frizzled, evolved from the
Dictyostelium discoideum cAMP receptors before the split of Opisthokonts. Later, the
Secretin family evolved from the
Adhesion GPCR receptor family before the split of
nematodes.