Lecture 2: G-Coupled Protein Receptors Flashcards
1
Q
What is the structure of a GPCR?
A
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.
2
Q
To what extent do drugs target GCPRs?
A
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.
3
Q
How many GPCRs are there in humans compared to mice?
A
There are more than 800 GPCRs in humans, and in mice there are about 1000 concerned with the sense of smell alone.
4
Q
What general effect does activation have on a GPCR?
A
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
5
Q
When does the G-protein bind to the receptor?
A
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.
6
Q
How varied are G-proteins?
A
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.
7
Q
What forms the G-protein coupled to the receptor?
A
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 .
8
Q
How is the G protein relevant to the function of the receptor?
A
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
9
Q
Describe how these subunits functionally compose an inactive GPCR
A
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.
10
Q
What is the relevance of the α subunit acting as a GTPase?
A
The α subunit is a GTPase and becomes inactive when it hydrolyzes its bound GTP to GDP.
11
Q
How long does it take for GTP hydrolysis to occur and why is this?
A
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).
12
Q
What are the function of these regulators of g-protein signalling?
A
RGS proteins act as α-subunit-specific GTPase-activating proteins (GAPs), and they help shut off G-protein-mediated responses in all eukaryotes.
13
Q
Name two important domains of the α subunit of the G protein
A
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
14
Q
How is the AH binding domain relevant to its function?
A
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
15
Q
What is the function of cAMP?
A
Cyclic AMP (cAMP) acts as a second messenger in some signaling pathways.
16
Q
An extracellular signal can increase cAMP concentration more than twentyfold in seconds. What is required for this?
A
Such a rapid response requires balancing a rapid synthesis of the molecule with its rapid breakdown or removal.
17
Q
How is cAMP synthesised and degraded?
A
Cyclic AMP is synthesised from ATP by an enzyme called adenylyl cyclase, and it is rapidly and continuously destroyed by cyclic AMP phosphodiesterases
18
Q
Describe the structure of adenylyl cyclase
A
Adenylyl cyclase is a large, multipass transmembrane protein with its catalytic domain on the cytosolic side of the plasma membrane.
19
Q
What is the relevance of GPCRs to cAMP?
A
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.
20
Q
How are Gs and Gi medically relevant?
A
Both Gs and Gi are targets for medically important bacterial toxins.
21
Q
What toxin is relevant to Gs?
A
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.
22
Q
What toxin is relevant to Gi?
A
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.
23
Q
How are these toxins relevant in research
A
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.
24
Q
How do different cell types respond differently to an increase in cAMP concentration?
A
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.
25
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.
26
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).
27
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.
28
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
29
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.
30
What other function can AKAPs have?
Some AKAPs also bind other signaling proteins, forming a signaling complex.
31
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.
32
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.
33
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.
34
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.
35
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.
36
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
37
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.
38
What similarities do these two PKA subtypes show when activated?
In both types, once the catalytic subunits are freed and active, they can migrate into the nucleus (where they can phosphorylate transcription regulatory proteins), while the regulatory subunits remain in the cytoplasm.
39
Therefore describe hollistically how a rise in intracellular cyclic AMP concentration can alter gene transcription.
The binding of an extracellular signal molecule to its GPCR activates adenylyl cyclase via Gs and thereby increases cAMP concentration in the cytosol. This rise activates PKA, and the released catalytic subunits of PKA
can then enter the nucleus, where they phosphorylate the transcription regulatory protein CREB. Once phosphorylated, CREB recruits the coactivator CBP, which stimulates gene transcription. In some cases, at least, the inactive CREB protein is bound to the cyclic AMP response element (CRE) in DNA before it is phosphorylated
40
Name 2 family members, the subunits that mediate their action and their functions of the family I of trimeric G proteins
G.s: α: Activates adenylyl cyclase; activates Ca2+ channels
G.olf: α: Activates adenylyl cyclase in olfactory sensory neurons
41
Name 3 family members, the subunits that mediate their action and their functions of the family II of trimeric G proteins
G.i: α: Inhibits adenylyl cyclase
βγ: Activates K+ channels
G.o: βγ: Activates K+ channels; inactivates Ca2+ channels
α + βγ: Activates phospholipase C-β
G.t: α: Activates cyclic GMP phosphodiesterase in vertebrate rod photoreceptors (transducin)
42
Name a family member, the subunits that mediate its action and their functions of the family III of trimeric G proteins
G.q: α: Activates phospholipase C-β
43
Name a family member, the subunits that mediate its action and their functions of the family IV of trimeric G proteins
G.12/13: α: Activates Rho family monomeric GTPases (via Rho-GEF) to regulate the actin cytoskeleton
44
How can G protein-mediated signalling by agonist-activated GPCRs be terminated?
G protein-mediated signalling by agonist-activated GPCRs can be terminated through GPCR phosphorylation by GPCR kinases (GRKs) and concomitant GPCR association with arrestins via these phosphorylation sites, which interact with clathrin and the clathrin adaptor AP2 to drive GPCR internalization into endosomes in an almost clathrin lined pit.
45
What function may this termination have? How would this play out?
GPCR internalisation regulates the functional process of receptor desensitisation. Recruitment of arrestins to activated GPCRs can also lead to the initiation of distinct arrestin-mediated signalling pathways, including activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) pathway.
46
What is the fate of GPCRs when after they are trafficked into endosomes?
Following internalization after association with arrestins, GPCRs can be trafficked to lysosomes, where they are ultimately degraded, or to recycling endosomes for recycling back to the cell surface in the functional process of resensitization — whereby the cell is resensitized for another round of signalling.
47
What interesting finding has been made recently about 'biased' agonists?
Interestingly, 'biased' agonists have been recently characterised that specifically activate G protein-mediated signalling pathways over arrestin-mediated GPCR signalling pathways, or vice versa. This new concept illustrates the importance of characterizing all GPCR downstream signalling pathways in order to fully exploit the therapeutic potential of clinically important receptors.
48
Aside from perstussis and cholera toxin, name two ways in which you can interfere with G-protein mediated signaling
GTPgammaS: Keeps it active to allow study of effect
[(35)S]GTPgammaS: Can use this as a measure of activity.
49
Describe in more detail how GTPgammaS remains active
GTPgammaS is a non-hydrolyzable or slowly hydrolyzable G-protein-activating analog of guanosine triphosphate (GTP).
Many GTP binding proteins demonstrate activity when bound to GTP, and are inactivated via the hydrolysis of the phosphoanhydride bond that links the γ-phosphate to the remainder of the nucleotide, leaving a bound guanosine diphosphate (GDP) and releasing an inorganic phosphate. This usually occurs rapidly, and the GTP-binding protein can then only be activated by exchanging the GDP for a new GTP molecule.
The substitution of sulfur for one of the oxygens of the γ-phosphate of GTP creates a nucleotide that either cannot be hydrolyzed or is only slowly hydrolyzed. This prevents the GTP-binding proteins from being inactivated, and allows the cellular processes that they carry out when active to be more easily studied.
50
Describe in more detail how [35S]GTPgammaS can be used as a measure of activity
You can try to activate it with compounds and use this to see if it binds (radioactivity). This can be done to find a novel ligand of a receptor. The GTPgamma keeps it active. Because of the gammaS it won’t be hydrolysed, you don’t lose the radioactivity. You can do this with a high number of receptors and modify a ligand to make it more effective.
51
What membrane bound enzyme do many GPCRs exert their effects through?
Many GPCRs exert their effects through G proteins that activate the plasmmem- brane-bound enzyme phospholipase C-β (PLCβ).
52
What is the immediate effect of phospholipase C-β activation?
The phospholipase acts on a phosphorylated inositol phospholipid (a phosphoinositide) called phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]; PIP2, which is present in small amounts in the inner half of the plasma membrane lipid bilayer
The activated phospholipase then cleaves the PI(4,5)P2 to generate two products: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. At this step, the signaling pathway splits into two branches.
53
Which G protein is often coupled to receptors that activate this inositol phospholipid signaling pathway?
Receptors that activate this inositol phospholipid signaling pathway mainly do so via a G protein called Gq, which activates phospholipase C-β in much the same way that Gs activates adenylyl cyclase.
54
Describe the first branch of this pathway involving IP3
IP3 is a water-soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol.
When it reaches the endoplasmic reticulum (ER), it binds to and opens IP3-gated Ca2+-release channels (also called IP3 receptors) in the ER membrane.
Ca2+ stored in the ER is released through the open channels, quickly raising the concentration of Ca2+ in the cytosol. The increase in cytosolic Ca2+ propagates the signal by influencing the activity of Ca2+- sensitive intracellular proteins.
55
Describe the other branch of this pathway involving IP3 diacylglycerol
At the same time, diacylglycerol also acts as a second messenger, but it remains embedded in the plasma membrane, where it has several potential signalling roles. One of its major functions is to activate a protein kinase called protein kinase C (PKC), so named because it is Ca2+-dependent. The initial rise in cytosolic Ca2+ induced by IP3 alters the PKC so that it translocates from the cytosol to the cytoplasmic face of the plasma membrane. There it is activated by the combination of Ca2+, diacylglycerol, and the negatively charged membrane phospholipid phosphatidylserine
56
How can this Gq-PIP2 pathway evoke inflammatory responses downstream? What is the clinical relevance of this?
Diacylglycerol can be further cleaved to release arachidonic acid, which can either act as a signal in its own right or be used in the synthesis of other small lipid signal molecules called eicosanoids. Most vertebrate cell types make eicosanoids, including prostaglandins, which have many biological activities. They participate in pain and inflammatory responses, for example, and many anti-inflammatory drugs (such as aspirin, ibuprofen, and cortisone) act in part by inhibiting their synthesis.
57
How are G proteins related to dopamine receptor families
D1 is a Gs and D2 is a Gi
58
Humans can distinguish more than 10,000 distinct smells, which they detect using specialized olfactory receptor neurons in the lining of the nose. What names are given to these receptors?
These cells use specific GPCRs called olfactory receptors to recognize odors; the receptors are displayed on the surface of the modified cilia that extend from each cell
59
What signalling molecules do these olfactory receptors act through? What are the downstream effects of this?
The receptors act through cAMP. When stimulated by odorant binding, they activate an olfactory-specific G protein (known as Golf), which in turn activates adenylyl cyclase. The resulting increase in cAMP opens cyclic-AMP-gated cation channels, thereby allowing an influx of Na+, which depolarizes the olfac- tory receptor neuron and initiates a nerve impulse that travels along its axon to the brain.
60
How many olfactory receptors are there, how varied are they and how many are in each neuron?
There are about 1000 different olfactory receptors in a mouse and about 350 in a human, each encoded by a different gene and each recognizing a different set of odorants. Each olfactory receptor neuron produces only one of these receptors
The neuron responds to a specific set of odorants by means of the specific recep- tor it displays, and each odorant activates its own characteristic set of olfactory receptor neurons
61
How do these receptors aid in development?
The same receptor also helps direct the elongating axon of each developing olfactory neuron to the specific target neurons that it will connect to in the brain.
62
How do a different set of receptors carry out a similar function and are thought to be lacking in humans?
A different set of GPCRs acts in a similar way in some vertebrates to mediate responses to pheromones, chemical signals detected in a different part of the nose that are used in communication between members of the same species. Humans, however, are thought to lack functional pheromone receptors.
63
How do the receptors for vision differ to that of olfaction?
Vertebrate vision employs a similarly elaborate, highly sensitive, signal-detection process. Cyclic-nucleotide-gated ion channels are also involved, but the crucial cyclic nucleotide is cyclic GMP rather than cAMP.
In visual transduction responses, which are the fastest G-protein-mediated responses known in vertebrates, the receptor activation stimulated by light causes a fall rather than a rise in the level of the cyclic nucleotide.
64
Describe the synthesis and degredation of GMP
As with cAMP, a continuous rapid synthesis (by guanylyl cyclase) and rapid degradation (by cyclic GMP phosphodiesterase) controls the concentration of cyclic GMP in the cytosol.
65
What encodes colour and noncolour vision, and which is the better studied receptor?
The pathway has been especially well studied in rod photoreceptors (rods) in the vertebrate retina. Rods are responsible for noncolor vision in dim light, whereas cone photoreceptors (cones) are responsible for color vision in bright light.
66
Describe the structure of rod receptors
A rod photoreceptor is a highly specialised cell with outer and inner segments, a cell body, and a synaptic region where the rod passes a chemical signal to a retinal nerve cell
67
Where is the phototransduction apparatus in the rod photoreceptor?
The phototransduction apparatus is in the outer segment of the rod, which contains a stack of discs, each formed by a closed sac of membrane that is densely packed with photosensitive rhodopsin molecules. The plasma membrane surrounding the outer segment contains cyclic-GMP-gated cation channels. Cyclic GMP bound to these channels keeps them open in the dark.
68
How does depolarisation and hyperpolarisation occur in these rod photoreceptors?
Paradoxically, light causes a hyperpolarisation rather than a depolarisation of the plasma membrane. Hyperpolarisation results because the light-induced activation of rhodopsin molecules in the disc membrane decreases the cyclic GMP concentration and closes the cation channels in the surrounding plasma membrane
69
Describe rhodopsins and their effects once activated
Rhodopsin is an integral protein with seven membrane-spanning a helices, the characteristic GPCR architecture, but the activating extracellular signal is not a molecule but a photon of light. 11-cis-retinal (a chromophore) is covalently attached to opsin, the protein component of rhodopsin,, which isomerises almost instantaneously to all-trans retinal when it absorbs a single photon.
70
What downstream effects does this isomerisation of retinal have
The isomerisation alters the shape of the retinal, forcing a conformational change in the protein (opsin). The activated rhodopsin molecule then alters the conformation of the G protein transducin (Gt), causing the transducin α subunit to activate cyclic GMP phosphodiesterase. The phosphodiesterase then hydrolyzes cyclic GMP, so that cyclic GMP levels in the cytosol fall. This drop in cyclic GMP concentration decreases the amount of cyclic GMP bound to the plasma membrane cation channels, allowing more of these cyclic-GMP-sensitive channels to close.
In this way, the signal quickly passes from the disc membrane to the plasma membrane, and a light signal is converted into an electrical one, through a hyperpolarization of the rod cell plasma membrane.
71
What kind of feedback is present in rod photoreceptors? Why is this necessary?
Rods use several negative feedback loops to allow the cells to revert quickly to a resting, dark state in the aftermath of a flash of light—a requirement for perceiving the shortness of the flash.
72
Describe two ways in which this negative feedback can work in rod photoreceptors
A rhodopsin-specific protein kinase called rhodopsin kinase (RK) phosphorylates the cytosolic tail of activated rhodopsin on multiple serines, partially inhibiting the ability of the rhodopsin to activate transducin. An inhibitory protein called arrestin then binds to the phosphorylated rhodopsin, further inhibiting rhodopsin’s activity. Mice or humans with a mutation that inactivates the gene encoding RK have a prolonged light response.
At the same time as arrestin shuts off rhodopsin, an RGS protein binds to activated transducin, stimulating the transducin to hydrolyze its bound GTP to GDP, which returns transducin to its inactive state.
73
How do rod photoreceptors return to a ready state quickly?
The cation channels that close in response to light are permeable to Ca2+, as well as to Na+, so that when they close, the normal influx of Ca2+ is inhibited, causing the Ca2+ concentration in the cytosol to fall. The decrease in Ca2+ concentration stimulates guanylyl cyclase to replenish the cyclic GMP, rapidly returning its level to where it was before the light was switched on.
A specific Ca2+-sensitive protein mediates the activation of guanylyl cyclase in response to the fall in Ca2+ levels. In contrast to calmodulin, this protein is inactive when Ca2+ is bound to it and active when it is Ca2+-free. It therefore stimulates the cyclase when Ca2+ levels fall following a light response.
74
Aside from allowing the rod to return to its resting state, what else do these negative feedback mechanisms do?
Negative feedback mechanisms do more than just return the rod to its resting state after a transient light flash; they also help the rod to adapt, stepping down the response when the rod is exposed to light continuously. Adaptation, as we discussed earlier, allows the receptor cell to function as a sensitive detector of changes in stimulus intensity over an enormously wide range of baseline levels of stimulation. It is why we can see faint stars in a dark sky, or a camera flash in bright sunlight.
75
Where are rods and cones placed relevant to the rest of the retina?
The lens focuses light on the retina, which is composed of layers of neurons. The primary photosensory neurons are rod cells, which are responsible for high-resolution and night vision, and cone cells of three subtypes, which initiate colour vision. The rods and cones form synapses with several ranks of interconnecting neurons that convey and integrate the electrical signals. The signals eventually pass from ganglion neurons through the optic nerve to the brain. Note that light must pass through the layers of ganglion neurons and interconnecting neurons before reaching the rod and cone cells.
76
Where does the retinal lie in the receptor?
The chromophore 11-cis-retinal, attached through a Schiff base linkage to Lys256 of the seventh helix, lies near the center of the bilayer. It is oriented with its long axis approximately in the plane of the membrane.
77
What is retinal derived from?
Retinal is derived from vitamin A1 (retinol),
which is produced from B-carotene. Dietary deficiency of vitamin A leads to night blindness (the inability to adapt to low light levels), which is relatively common in some developing countries.
78
Describe how rhodopsin is bound to the G protein
Rhodopsin is palmitoylated (bound to a fatty acid) at its carboxyl terminus, and both the a and y subunits of transducin have attached lipids (yellow) that assist in anchoring them to the membrane.
79
Describe how cGMP changes the transmembrane potential
The rod cell consists of an outer segment, filled with stacks of membranous disks containing the photoreceptor rhodopsin, and an inner segment that contains the nucleus and other organelles. The inner segment forms a synapse with interconnecting neurons. Cones have a similar structure.
ATP in the inner segment powers the Na+K+ ATPase, which creates a transmembrane electrical potential by pumping 3 Na+ out for every 2 K+ pumped in. The membrane potential is reduced by the inflow of Na+ and Ca2+ through cGMP-gated cation channels in the outer-segment plasma membrane.
When rhodopsin absorbs light, it triggers degradation of cGMP in the outer segment, causing closure of the ion channel. Without cation influx through this channel, the cell becomes hyperpolarised. This electrical signal is passed to the brain through the ranks of neurons
80
How similar is transducin to Gi and Gs?
Transducin (T) belongs to the same family of heterotrimeric GTP-binding proteins as Gs and Gi. Although specialised for visual transduction, transducin shares many functional features with Gs and Gi. It can bind either GDP or GTP.
81
Describe how GDP and GTP interact with transducin
In the dark, GDP is bound, all three subunits of the protein (Ta, TB, and Ty) remain together, and no signal is sent. When rhodopsin is excited by light, it interacts with transducin, catalysing the replacement of bound GDP by GTP from the cytosol
82
What happens to transducin following excitation?
Transducin then dissociates into Ta and TBy, and the Ta-GTP carries the signal from the excited receptor to the next element in the transduction pathway, a cGMP phosphodiesterase; this enzyme converts cGMP to 5'-GMP
83
Where does PDE fit into this pathway?
One isoform of the cGMP-specific PDE is unique to the visual cells of the retina. The PDE of the retina is a peripheral protein with its active site on the cytoplasmic side of the disk membrane.
84
How does PDE and the G protein subunits interact?
In the dark, a tightly bound inhibitory subunit very effectively suppresses the PDE activity. When Ta- GTP encounters the PDE, the inhibitory subunit leaves the enzyme and instead binds Ta, and the enzyme’s activity immediately increases by several orders of magnitude.
85
What are the consequences of this actuvation of the PDE?
Each molecule of the active PDE degrades many molecules of cGMP to the biologically inactive 5-GMP, lowering [cGMP] in the outer segment within a fraction of a second. At the new, lower [cGMP], the cGMP-gated ion channels close, blocking reentry of Na+ and Ca2+ into the outer segment and hyperpolarising the membrane of the rod or cone cell. Through this process, the initial stimulus—a photon—changes the Vm of the cell.
The brighter the illumination of the rod cell, the greater the hyperpolarisation. This hyperpolarisation is perceived by the integrating neurons of the retina, which pass the integrated signal on to the ganglion cells, which send axons via the optic nerve to the brain.
86
Several steps in the visual-transduction process result in a huge amplification of the signal. Describe this
Each excited rhodopsin molecule activates at least 500 molecules of transducin, each of which can activate a molecule of the PDE.
This phosphodiesterase has a remarkably high turnover number, each activated molecule hydrolyzing 4,200 molecules of cGMP per second.
The binding of cGMP to cGMP-gated ion channels is cooperative, and a relatively small change in [cGMP] therefore registers as a large change in ion conductance.
The result of these amplifications is exquisite sensitivity to light. Absorption of a single photon closes 1,000 or more ion channels and changes the cell’s membrane potential by about 1 mV.
87
As your eyes move across this line, the retinal images of the first words disappear rapidly—before you see the next series of words. What allows for this?
Very shortly after illumination of the rod or cone cells stops, the photosensory system shuts off. The a subunit of transducin (with bound GTP) has intrinsic GTPase activity. Within milliseconds after the decrease in light intensity, GTP is hydrolyzed and Ta reassociates with TBy. The inhibitory subunit of the PDE, which had been bound to Ta-GTP, is released and reassociates with the enzyme, strongly inhibiting its activity.
88
To return [cGMP] to its “dark” level, what happens?
The enzyme
guanylyl cyclase converts GTP to cGMP, in a reaction that is inhibited by high [Ca2+]. Calcium levels drop during illumination, because the steady-state [Ca2+] in the outer segment is the result of outward pumping of Ca+ through the Na+ - Ca2+ exchanger of the plasma membrane and influx of Ca2+ through open cGMP-gated channels. In the dark, this produces a [Ca2+] of about 500 nM— enough to inhibit cGMP synthesis. After brief illumination, Ca2+ entry slows and [Ca2+] declines. The inhibition of guanylyl cyclase by Ca2+ is relieved, and the cyclase converts GTP to cGMP to return the system to its prestimulus state
89
How does rhodopsin react to prolonged illumination
Rhodopsin itself also undergoes changes in response to prolonged illumination. The conformational change
induced by light absorption exposes several Thr and Ser residues in the carboxyl-terminal domain. These residues are quickly phosphorylated by rhodopsin kinase which is functionally and structurally homologous to the B-adrenergic kinase (BARK) that desensitizes the B-adrenergic receptor
90
What other proteins are involved in this inhibition of rhodopsin?
The Ca2+ binding protein recoverin inhibits rhodopsin kinase at high [Ca2+], but the inhibition is relieved when [Ca2+] drops after illumination. The phosphorylated carboxyl-terminal domain of rhodopsin is bound by the protein arrestin 1 (apparently same as recoverin), preventing further interaction between activated rhodopsin and transducin.
91
Describe what happens to retinal during this desensitisation process
On a relatively long time scale (seconds to min- utes), the all-trans-retinal of an excited rhodopsin molecule is removed and replaced by 11-cis-retinal, to produce rhodopsin that is ready for another round of excitation
92
So hollistically, give 9 steps which describe the molecular consequences of photon absorption by rhodopsin in the rod outer segment.
1. Light absorption converts 11-cis- retinal to all-trans- retinal, activating rhodopsin (Rh).
2. Activated rhodopsin catalyzes replacement of GDP by GTP on transducin (T), which then dissociates into Ta-GTP and TBy.
3. Ta-GTP activates cGMP phosphodiesterase (PDE) by binding and removing its inhibitory subunit (I).
4. Active PDE reduces [cGMP] to below the level needed to keep cation channels open.
5. Cation channels close, preventing influx of Na+ and Ca2+; membrane is hyperpolarized. This signal passes to the brain.
6. Continued eflux of Ca2+ through the Na+-Ca2+ exchanger reduces cytosolic [Ca2+].
7. Reduction of [Ca2+] activates guanylyl cyclase (GC) and inhibits PDE; [cGMP] rises toward “dark” level, reopening cation channels and returning Vm to prestimulus level.
8. Rhodopsin kinase (RK) phosphorylates “bleached” rhodopsin; low [Ca2+]
and recoverin (Recov) stimulate this reaction. Arrestin (Arr) binds phosphorylated carboxyl terminus, inactivating rhodopsin.
9. Slowly, Arrestin dissociates, rhodopsin is dephosphorylated, and all-trans- retinal is replaced with 11-cis-retinal. Rhodopsin is ready for another phototransduction cycle.
93
Describe a better alternative to CO-IP
Proximity ligation assay:
1. Incubate with target primary antibodies from two different species
2. Add PLA probes plus and minus
3. Hybridise connector oligos
4. Ligation to form a complete DNA circle
5. Rolling circle amplification
6. Add flourescent probes to reveal phosphorylation
94
Why might PLA be better than CO-IP?
One interaction and way more sensitive. Cells must be dead however can be done in the cell in which you are interested without lysating the cell. Can look whether in the nucleus or in the cytoplasm. You want to show that transduction pathways are present in the organism in the area you are interested in and not just in a dish.
