Lecture 2: G-Coupled Protein Receptors Flashcards
What is the structure of a GPCR?
They consist of a single polypeptide chain that threads back and forth across the lipid bilayer seven times, forming a cylindrical structure, often with a deep ligand-binding site at its center. In addition to their characteristic orientation in the plasma membrane, they all use G proteins to relay the signal into the cell interior.
To what extent do drugs target GCPRs?
It is remarkable that almost half of all known drugs work through GPCRs or the signaling pathways GPCRs activate. Of the many hundreds of genes in the human genome that encode GPCRs, about 150 encode orphan receptors, for which the ligand is unknown.
How many GPCRs are there in humans compared to mice?
There are more than 800 GPCRs in humans, and in mice there are about 1000 concerned with the sense of smell alone.
What general effect does activation have on a GPCR?
Activation results in a conformational change it its intracellular loop that enables it to activate a trimeric GTP-binding protein (G protein), which couples the receptor to enzymes or ion channels in the membrane. Causes the tail to undergo a conformational change and release the subunits. GTP has a higher energy state due to having an extra phosphor
When does the G-protein bind to the receptor?
In some cases, the G protein is physically associated with the receptor before the receptor is activated, whereas in others it binds only after receptor activation.
How varied are G-proteins?
There are various types of G proteins, each specific for a particular set of GPCRs and for a particular set of target proteins in the plasma membrane. They all have a similar structure, however, and operate similarly.
What forms the G-protein coupled to the receptor?
G proteins are composed of three protein subunits—α, β, and γ. In the unstimulated state, the α subunit has GDP bound and the G protein is inactive .
How is the G protein relevant to the function of the receptor?
When a GPCR is activated, it acts like a guanine nucleotide exchange factor (GEF) and induces the α subunit to release its bound GDP, allowing GTP to bind in its place. GTP binding then causes an activating conformational change in the Gα subunit, releasing the G protein from the receptor and triggering dissociation of the GTP-bound Gα subunit from the Gβγ pair—both of which then interact with various targets, such as enzymes and ion channels in the plasma membrane, which relay the signal onward
Describe how these subunits functionally compose an inactive GPCR
Both the α and the γ subunits have covalently attached lipid molecules (two amino acid chains) that help bind them to the plasma membrane, and the α subunit has GDP bound.
The α subunit contains the GTPase domain and binds to one side of the β subunit. The γ subunit binds to
the opposite side of the β subunit, and the β and γ subunits together form a single functional unit.
What is the relevance of the α subunit acting as a GTPase?
The α subunit is a GTPase and becomes inactive when it hydrolyzes its bound GTP to GDP.
How long does it take for GTP hydrolysis to occur and why is this?
The time required for GTP hydrolysis is usually short because the GTPase activity is greatly enhanced by the binding of the α subunit to a second protein, which can be either the target protein or a specific regulator of G protein signaling (RGS).
What are the function of these regulators of g-protein signalling?
RGS proteins act as α-subunit-specific GTPase-activating proteins (GAPs), and they help shut off G-protein-mediated responses in all eukaryotes.
Name two important domains of the α subunit of the G protein
The GTPase domain of the α subunit contains two major subdomains: the “Ras” domain, which is related to other GTPases and provides one face of the nucleotide-binding pocket; and the alpha- helical or “AH” domain, which clamps the nucleotide in place
How is the AH binding domain relevant to its function?
Binding of an extracellular signal molecule to a GPCR changes the conformation of the receptor, which allows the receptor to bind and alter the conformation of a trimeric G protein.
The AH domain of the G protein α subunit moves outward to open the nucleotide-binding site, thereby promoting dissociation of GDP. GTP binding then promotes closure of the nucleotide-binding site, triggering conformational changes that cause dissociation of the α subunit from the receptor and from the βγ complex.
The GTP-bound α subunit and the βγ complex each regulate the activities of downstream signaling molecules. The receptor stays active while the extracellular signal molecule is bound to it, and it can therefore catalyse the activation of many G-protein molecules
What is the function of cAMP?
Cyclic AMP (cAMP) acts as a second messenger in some signaling pathways.
An extracellular signal can increase cAMP concentration more than twentyfold in seconds. What is required for this?
Such a rapid response requires balancing a rapid synthesis of the molecule with its rapid breakdown or removal.
How is cAMP synthesised and degraded?
Cyclic AMP is synthesised from ATP by an enzyme called adenylyl cyclase, and it is rapidly and continuously destroyed by cyclic AMP phosphodiesterases
Describe the structure of adenylyl cyclase
Adenylyl cyclase is a large, multipass transmembrane protein with its catalytic domain on the cytosolic side of the plasma membrane.
What is the relevance of GPCRs to cAMP?
Many extracellular signals work by increasing cAMP concentrations inside the cell. These signals activate GPCRs that are coupled to a stimulatory G protein (Gs). The activated α subunit of Gs binds and thereby activates adenylyl cyclase. Other extracellular signals, acting through different GPCRs, reduce cAMP levels by activating an inhibitory G protein (Gi), which then inhibits adenylyl cyclase.
How are Gs and Gi medically relevant?
Both Gs and Gi are targets for medically important bacterial toxins.
What toxin is relevant to Gs?
Cholera toxin, which is produced by the bacterium that causes cholera, is an enzyme that catalyzes the transfer of ADP ribose from intracellular NAD+ to the α subunit of Gs. This ADP ribosylation alters the α subunit so that it can no longer hydrolyze its bound GTP, causing it to remain in an active state that stimulates adenylyl cyclase indefinitely. The resulting prolonged elevation in cAMP concentration within intestinal epithelial cells causes a large efflux of Cl– and water into the gut, thereby causing the severe diarrhoea that characterises cholera.
What toxin is relevant to Gi?
Pertussis toxin, which is made by the bacterium that causes pertussis (whooping cough), catalyzes the ADP ribosylation of the α subunit of Gi, preventing the protein from interacting with receptors; as a result, the G protein remains in the inactive GDP-bound state and is unable to regulate its target proteins.
How are these toxins relevant in research
These two toxins are widely used in experiments to determine whether a cell’s GPCR-dependent response to a signal is mediated by Gs or by Gi.
How do different cell types respond differently to an increase in cAMP concentration?
Some cell types, such as fat cells, activate adenylyl cyclase in response to multiple hormones, all of which thereby stimulate the breakdown of triglyceride (the storage form of fat) to fatty acids.
What can occur from genetic defects to the Gs a subunit?
Individuals with genetic defects in the Gs α subunit show decreased responses to certain hormones, resulting in metabolic abnormalities, abnormal bone development, and mental retardation.
How does cAMP exert its effects in most animal cells?
In most animal cells, cAMP exerts its effects mainly by activating cyclic-AMP- dependent protein kinase (PKA).
What effect does PKA have?
This kinase phosphorylates specific serines or threonines on selected target proteins, including intracellular signaling proteins and effector proteins, thereby regulating their activity. The target proteins differ from one cell type to another, which explains why the effects of cAMP vary so markedly depending on the cell type.
What effect does cAMP have on PKA?
In the inactive state, PKA consists of a complex of two catalytic subunits and two regulatory subunits. The binding of cAMP to the regulatory subunits alters their conformation, causing them to dissociate from the complex. The released catalytic subunits are thereby activated to phosphorylate specific target proteins
Why are the regulatory subunits of PKA important?
The regulatory subunits of PKA (also called A-kinase) are important for localising the kinase inside the cell: special A-kinase anchoring proteins (AKAPs) bind both to the regulatory subunits and to a component of the cytoskeleton or a membrane of an organelle, thereby tethering the enzyme complex to a particular subcellular compartment.
What other function can AKAPs have?
Some AKAPs also bind other signaling proteins, forming a signaling complex.
Give an example of an AKAP mediated signalling complex in the heart and the function it performs
An AKAP located around the nucleus of heart muscle cells, for example, binds both PKA and a phosphodiesterase that hydrolyzes cAMP.
In unstimulated cells, the phosphodiesterase keeps the local cAMP concentration low, so that the bound PKA is inactive; in stimulated cells, cAMP concentration rapidly rises, overwhelming the phosphodiesterase and activating the PKA.
Among the target proteins that PKA phosphorylates and activates in these cells is the adjacent phosphodiesterase, which rapidly lowers the cAMP concentration again. This negative feedback arrangement converts what might otherwise be a prolonged PKA response into a brief, local pulse of PKA activity.
How can the timing of cAMP vary
Whereas some responses mediated by cAMP occur within seconds, others depend on changes in the transcription of specific genes and take hours to develop fully.
Give an example of the transcriptional effects of cAMP
In cells that secrete the peptide hormone somatostatin, for example, cAMP activates the gene that encodes this hormone.
Describe how cAMP regulates the transcription of somatostatin
The regulatory region of the somatostatin gene contains a short cis-regulatory sequence, called the cyclic AMP response element (CRE), which is also found in the regulatory region of many other genes activated by cAMP.
A specific transcription regulator called CRE-binding (CREB) protein recognises this sequence. When PKA is activated by cAMP, it phosphorylates CREB on a single serine; phosphorylated CREB then recruits a transcriptional coactivator called CREB-binding protein (CBP), which stimulates the transcription of the target genes.
What function is CREB thought to play in the brain?
Thus, CREB can transform a short cAMP signal into a long-term change in a cell, a process that, in the brain, is thought to play an important part in some forms of learning and memory.
How many cAMP molecules are required to activate PKA? What relevance does this have?
The release of the catalytic subunits requires the binding of more than two cAMP molecules to the regulatory subunits in the tetramer. This requirement greatly sharpens the response of the kinase to changes in cAMP concentration
What are the subtypes of mammalian PKA?
Mammalian cells have at least two types of PKAs: type I is mainly in the cytosol, whereas type II is bound via its regulatory subunits and special anchoring proteins to the plasma membrane, nuclear membrane, mitochondrial outer membrane, and microtubules.
