Biochem Chpt 12.2 GPCRs and second messengers

Question 1

What does the name GPCR tell regarding their behaviour?

Answer 1

As their name implies, G protein–coupled receptors (GPCRs) are receptors that are closely associated with a member of the guanosine nucleotide–binding protein (G protein) family.

Question 2

What three essential components define signal transduction through GPCRs?

Answer 2

• Plasma membrane receptor with seven transmembrane helical segments

*G protein that cycles between active (GTP- bound) and inactive (GDP-bound) forms

• An effector enzyme (or ion channel) in the plasma membrane that is regulated by the activated G protein.

Question 3

What happens when the G protein is stimulated?

Answer 3

The G protein, stimulated by the activated receptor, exchanges bound GDP for GTP, then dissociates from the occupied receptor and binds to the nearby effector enzyme, altering its activity. The activated enzyme then generates a second messenger that affects downstream targets

Question 4

How many GCPRs are encoded in the human genome?

Answer 4

The human genome encodes about 350 GPCRs for detecting hormones, growth factors, and other endogenous ligands, and perhaps 500 that serve as olfactory (smell) and gustatory (taste) receptors.

Question 5

What is the clinical relevance of GCPRs?

Answer 5

GPCRs have been implicated in many common human diseases, including allergies, depression, blindness, diabetes, and various cardiovascular defects with serious health consequences. Close to half of all drugs on the market target one GPCR or another.

For example, the B-adrenergic receptor, which mediates the effects of epinephrine, is the target of the “beta blockers,” prescribed for such diverse conditions as hypertension, cardiac arrhythmia, glaucoma, anxiety, and migraine headache.

Question 6

To what extent have these receptors been identified?

Answer 6

At least 150 of the GPCRs found in the human genome are still “orphan receptors”: their natural ligands are not yet identified, and so we know nothing about their biology.

Question 7

What is the function of epinephrine?

Answer 7

Epinephrine sounds the alarm when some threat requires the organism to mobilise its energy-generating machinery; it signals the need to fight or flee.

Question 8

What binds to what for epinephrine to function?

Answer 8

Epinephrine action begins when the hormone binds to a protein receptor in the plasma membrane of an epinephrine- sensitive cell. Adrenergic receptors (“adrenergic” reflects the alternative name for epinephrine, adrenaline) are of four general types, a1, a2, B1, and B2, defined by differences in their affinities and responses to a group of agonists and antagonists.

Question 9

What are meant by agonists and antagonists?

Answer 9

Agonists are structural analogs that bind to a receptor and mimic the effects of its natural ligand; antagonists are analogs that bind the receptor without triggering the normal effect and thereby block the effects of agonists, including the biological ligand. In some cases, the affinity of the synthetic agonist or antagonist for the receptor is greater than that of the natural agonist

Question 10

The four types of adrenergic receptors are found in different target tissues and mediate different responses to epinephrine. Where are the B-adrenergic receptors found? What do they do?

Answer 10

muscle, liver, and adipose tissue
These receptors mediate changes in fuel metabolism, including the increased breakdown of glycogen and fat.

Question 11

Why talk generally about the B-adrenergic receptors?

Answer 11

Adrenergic receptors of the B1 and B2 subtypes act through the same mechanism, so in our discussion, “B-adrenergic” applies to both types.

Question 12

Describe the B-adrenergic receptor

Answer 12

Like all GPCRs, the B-adrenergic receptor is an integral protein with seven hydrophobic, helical regions of 20 to 28 amino acid residues that span the plasma membrane seven times, thus the alternative name for GPCRs: heptahelical receptors.

Question 13

What name describes all G proteins which are involved in GPCRs?

Answer 13

For all GPCRs, the G protein is heterotrimeric, composed of three different subunits: a, B, and y (gamma). Such G proteins are therefore known as trimeric G proteins.

Question 14

Broadly, how does epinephrine affect the B-Adregenergic receptors?

Answer 14

The binding of epinephrine to a site on the receptor deep within the plasma membrane promotes a conformational change in the receptor’s intracellular domain that affects its interaction with an associated G protein, promoting the dissociation of GDP and the binding of GTP.

Question 15

Describe in regards to the individual subunits how this conformational effect of epinephrine on the B-adrenergic receptors take place

Answer 15

In this case, it is the a subunit that binds GDP or GTP and transmits the signal from the activated receptor to the effector protein. Because this G protein activates its effector, it is referred to as a stimulatory G protein, or Gs.

Like other G proteins, Gs functions as a biological “switch”: when the nucleotide-binding site of Gs (on the a subunit) is occupied by GTP, Gs is turned on and can activate its effector protein (adenylyl cyclase in the present case); with GDP bound to the site, Gs is switched off.

In the active form, the B and y subunits of Gs dissociate from the a subunit as a By dimer, and Gsa, with its bound GTP, moves in the plane of the membrane from the receptor to a nearby molecule of adenylyl cyclase. Gsa is held to the membrane by a covalently attached palmitoyl group

Question 16

What is adenylyl cyclase?

Answer 16

Adenylyl cyclase is an integral protein of the plasma membrane, with its active site on the cytoplasmic face.

Question 17

What does this association of adenylyl cyclase and Gsa incur?

Answer 17

The association of active Gsa with adenylyl cyclase stimulates the cyclase to catalyse cAMP synthesis from ATP, raising the cytosolic [cAMP]. The interaction between Gsa and adenylyl cyclase is possible only when Gsa is bound to GTP.

Question 18

How does Gsa binding of adrenylyl cyclase relate to its own activity?

Answer 18

The stimulation by Gsa is self-limiting; Gsa has intrinsic GTPase activity that inactivates Gsa by converting its bound GTP to GDP

Question 19

What happens to Gsa and adrenyl cyclase once it is inactive?

Answer 19

The now inactive Gsa dissociates from adenylyl cyclase, rendering the cyclase inactive. Gsa reassociates with the By dimer (GsBy), and inactive Gs is again available to interact with a hormone-bound receptor.

Question 20

What aspect of this function is reflective of a general role of G proteins?

Answer 20

The role of Gsa in serving as a biological “switch” protein is not unique. A variety of G proteins act as binary switches in signaling systems with GPCRs and in many processes that involve membrane fusion or fission

Question 21

How does epinephrine exert its downstream effects following this cascade?

Answer 21

Epinephrine exerts its downstream effects through the increase in [cAMP] that results from the activation of adenylyl cyclase. Cyclic AMP, in turn, allosterically activates cAMP-dependent protein kinase, also called protein kinase A or PKA which catalyses the phosphorylation of specific Ser or Thr residues of targeted proteins.

Example: glycogen phosphorylase b kinase. This enzyme is active when phosphorylated and can begin the process of mobilising glycogen stores in muscle and liver in anticipation of the need for energy, as signalled by epinephrine.

Question 22

Describe the structure of inactive PKA

Answer 22

The inactive form of PKA contains two identical catalytic subunits (C) and two identical regulatory sub- units (R). The tetrameric R2C2 complex is catalytically inactive, because an autoinhibitory domain of each R subunit occupies the substrate-binding cleft of each C subunit. aka the inhibitor sequences of the R subunits lie in the substrate-binding cleft of the C subunits and prevent binding of protein substrates. The amino-terminal sequences of the R subunits interact to form an R2 dimer, the site of binding to an A kinase anchoring protein (AKAP).

Question 23

How does this molecular cascade therefore affect PKA?

Answer 23

When [cAMP] rises in response to a hormonal signal, each R subunit binds two cAMP molecules and undergoes a dramatic reorganization that pulls its inhibitory sequence away from the C subunit, opening up the substrate-binding cleft and releasing each C subunit in its catalytically active form. The ATP- binding site of each catalytic subunit positions ATP perfectly for the transfer of its terminal (y) phosphoryl group to the —OH in the side chain of a Ser or Thr residue.

Question 24

How does PKA compare to other protein kinases? (2)

Answer 24

The structure of the substrate-binding cleft in PKA is the prototype for all known protein kinases; certain residues in this cleft region have identical counterparts in all of the more than 1,000 known protein kinases.

The same basic mechanism—displacement of an autoinhibitory domain— also mediates the allosteric activation of many types of protein kinases by their second messengers.

Question 25

What is the consequence of this activation of PKA?

Answer 25

PKA regulates several enzymes downstream in the signalling pathway. Although these downstream targets have diverse functions, they share a region of sequence similarity around the Ser or Thr residue that undergoes phosphorylation, a sequence that marks them for regulation by PKA. The substrate-binding cleft of PKA recognises these sequences and phosphorylates their Thr or Ser residue.

Question 26

What has the comparison of various protein substrates of PKA achieved?

Answer 26

Comparison of the sequences of various protein substrates for PKA has yielded the consensus sequence—the neighbouring residues needed to mark a Ser or Thr residue for phosphorylation.

Question 27

What has the comparison of various protein substrates of PKA achieved?

Answer 27

Comparison of the sequences of various protein substrates for PKA has yielded the consensus sequence—the neighbouring residues needed to mark a Ser or Thr residue for phosphorylation.

Question 28

As in many signaling pathways, signal transduction by adenylyl cyclase entails several steps that amplify the original hormone signal. Describe these steps

Answer 28

1. The binding of one hormone molecule to one receptor molecule catalytically activates many Gs molecules that associate with the activated receptor, one after the other. (10 molecules)
2. By activating one molecule of adenylyl cyclase, each active Gsa molecule stimulates the catalytic synthesis of many molecules of cAMP. (200 molecules)
3. The second messenger cAMP now activates PKA, each molecule (100 molecules) of which catalyses the phosphorylation of many molecules of the target protein: phosphorylase b kinase (1,000 molecules)
4. This kinase activates glycogen phosphorylase b (10,000), which leads to the rapid mobilization of glucose from glycogen. (100,000 molecules)

The net effect of the cascade is amplification of the hormonal signal by several orders of magnitude, which accounts for the very low concentration of epinephrine (or any other hormone) required for hormone activity.

Question 29

To be useful, a signal-transducing system has to turn off after the hormonal or other stimulus has ended. Describe two ways this can be achieved and name which B-adrenergic signalling demonstrates

Answer 29

Mechanisms for shutting off the signal are intrinsic to all signaling systems. Most systems also adapt to the continued presence of the signal by becoming less sensitive to it, in the process of desensitisation.

Question 30

What happens when the concentration of epinephrin in the blood drops below the Kd?

Answer 30

When the concentration of epinephrine in the blood drops below the Kd for its receptor, the hormone dissociates from the receptor and the latter reassumes the inactive conformation, in which it can no longer activate Gs.

Question 31

Describe another intrinsic means of ending the B-adrenergic response

Answer 31

A second means of ending the response to B-adrenergic stimulation is the hydrolysis of GTP bound to the Ga subunit, catalyzed by the intrinsic GTPase activity of the G protein. Conversion of bound GTP to GDP favors the return of Ga to the conformation in which it binds the GBy subunits—the conformation in which the G protein is unable to interact with or stimulate adenylyl cyclase. This ends the production of cAMP.

Question 32

What does the rate of inactivation of Gs depend on?

Answer 32

The rate of inactivation of Gs depends on the GTPase activity, which for Ga alone is very feeble. However, GTPase activator proteins (GAPs) strongly stimulate this GTPase activity, causing more rapid inactivation of the G protein

Question 33

How are GAP levels determined?

Answer 33

GAPs can themselves be regulated by other factors, providing a fine-tuning of the response to B-adrenergic stimulation.

Question 34

How may the response be terminated lower in the cascade? (2)

Answer 34

A third mechanism for terminating the response is to remove the sec- ond messenger: hydrolysis of cAMP to 5’-AMP (not active as a second messenger) by cyclic nucleotide phosphodiesterase

Finally, at the end of the signalling pathway, the metabolic effects that result from enzyme phosphorylaion are reversed by the action of phosphoprotein phosphatases, which hydrolyse phosphorylated Ser, Thr, or Tyr residues, releasing inorganic phosphate (Pi).

Question 35

How do the number of phosphoprotein phosphatases encoded by the genome compare to the number encoding protein kinases?

Answer 35

About 150 genes in the human genome encode phosphoprotein phosphatases, fewer than the number encoding protein kinases (,500). Some of these phosphatases are known to be regulated; others may act constitutively.

Question 36

What impact do these phosphoprotein phosphatases have in this cascade?

Answer 36

When [cAMP] drops and PKA returns to its inactive form, the balance between phosphorylation and dephosphorylation is tipped toward dephosphorylation by these phosphatases

Question 37

Thus, briefly give 7 stages in B-adrenergic signalling

Answer 37

1. Epinephrine binds to its
   specific receptor.
2. Hormone-receptor complex causes the GDP bound to Gsa to be replaced by GTP, activating
   Gsa.
3. Activated Gsa separates from GsBy, moves to adenylyl cyclase, and activates it. Many Gsa subunits may be activated by one occupied receptor.
4. Adenylyl cyclase catalyses the formation of cAMP.
5. cAMP activates PKA.
6. Phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine.
7. cAMP is degraded, reversing the activation of PKA.

Question 38

Describe the combined actions of stage 4 and 7 ()

Answer 38

Synthesis and hydrolysis of cAMP by adenylyl cyclase and cAMP phosphodiesterase, respectively.

4. In the presence of adenyl cyclase adenosine triohosphate (ATP) loses a PPi to form Adenosine 3’,5’-cyclic monophosphate (CAMP)
5. In the presence of cyclic nucleotide phosphodiesterase it gains H2o to become Adenosine 5’- monophosphate (AMP)

Question 39

Describe the Kd for two synthetic analogs of epinephrine

Answer 39

Epinephrines affinity for its receptor is expressed as a dissociation constant for the receptor-ligand complex (5). Isoproterenol and propranolol are synthetic analogs, one an agonist with an affinity for the receptor that is higher than that of epinephrine (0.4), and the other an antagonist with extremely high affinity (0.0046).

Question 40

How does the above mechanisms differ from desensitisation?

Answer 40

The mechanisms for signal termination described above take effect when the stimulus ends. A different mecha- nism, desensitization, damps the response even while the signal persists.

Question 41

How is the B-adrenergic receptor desensitised? (2)

Answer 41

Phosphorylation
Association with arrestin

Question 42

How is the B-adrenergic receptor phosphorylated?

Answer 42

Desensitisation of the B-adrenergic receptor is mediated by a protein kinase that phosphorylates the receptor on the intracellular domain that normally interacts with Gs. When the receptor is occupied by epinephrine, B-adrenergic receptor kinase, or BARK (also commonly called GRK2), phosphorylates several Ser residues near the carboxyl terminus of the receptor, which is on
the cytoplasmic side of the plasma membrane.

Question 43

How is BARK positioned to phosphorylate the receptor?

Answer 43

Usually located in the cytosol, BARK is drawn to the plasma membrane by its association with the GsBy subunits and is thus positioned to phosphorylate the receptor.

Question 44

How does this phosphorylation desensitise the receptor?

Answer 44

Receptor phosphorylation creates a binding site for the protein B-arrestin, or Barr (also called arrestin 2), and binding of B-arrestin effectively prevents further interaction between the receptor and the G protein.

Question 45

What further effect does the binding of B-arrestin have?

Answer 45

The binding of B-arrestin also facilitates receptor sequestration, the removal of receptor molecules from the plasma membrane by endocytosis into small intracellular vesicles.

Question 46

Describe how the system is re-sensitised

Answer 46

The arrestin-receptor complex recruits two proteins involved in vesicle formation, the AP-2 complex and clathrin, which initiate membrane invagination, leading to the formation of endosomes containing the adrenergic receptor. In this state the receptors are inaccessible to epinephrine and therefore inactive. Receptors in the endocytic vesicles are eventually dephosphorylated and returned to the plasma membrane, completing the circuit and re-sensitising the system to epinephrine

Question 47

To which family does BARK belong?

Answer 47

B-Adrenergic recep- tor kinase is a member of a family of G protein–cou- pled receptor kinases (GRKs), all of which phosphorylate GPCRs on their carboxyl-terminal cytoplasmic domains and play roles similar to that of BARK in desensitisation and resensitisation of their receptors.

Question 48

Comment on the numerocity of GRKs and arrestins encoded in the genome

Answer 48

At least five different GRKs and four different arrestins are encoded in the human genome; each GRK is capable of desensitising a particular subset of GPCRs, and each arrestin can interact with many different types of phosphorylated receptors.

Question 49

Name ten signals that use cAMP as a second messenger

Answer 49

Any of:
Corticotropin (ACTH)
Corticotropin-releasing hormone (CRH)
Dopamine [D1, D2]
Epinephrine (􏰀-adrenergic)
Follicle-stimulating hormone (FSH)
Glucagon
Histamine [H2]
Luteinizing hormone (LH)
Melanocyte-stimulating hormone (MSH)
Odorants (many)
Parathyroid hormone
Prostaglandins E1, E2 (PGE1, PGE2)
Serotonin [5-HT-1a, 5-HT-2]
Somatostatin
Tastants (sweet, bitter)
Thyroid-stimulating hormone (TSH)

Question 50

What common feature do all G proteins share which aids in their function?

Answer 50

They can become activated and then, after a brief period, can inactivate themselves, thereby serving as molecular binary switches with built-in timers. All G proteins have the same core structure and use the same mechanism for switching between an inactive conformation, favoured when GDP is bound, and an active conformation, favored when GTP is bound.

Question 51

Name 7 functions of the superfamily of G proteins and their associated proteins

Answer 51

Trimeric G proteins involved in adrenergic signalling (Gs & Gi)

Vision (Transducin)

Small G proteins such as that involved in insulin signaling (Ras)

Others that function in vesicle traficking (ARF & Rab)

Transport in and out of the nucleus (Ran)

Timing of the cell cycle (Rho)

Several proteins involved in protein synthesis (initiation factor IF2 and elongation factors EF-Tu and EF-G)

Question 52

Describe the GDP and GTP bound conformation of the G protein using the Ras protein, a minimal signaling unit, as a prototype for all members of this superfamily

Answer 52

In the GTP-bound conformation, the G protein exposes previously buried regions (called switch I and switch II) that interact with proteins down- stream in the signalling pathway, until the G protein inactivates itself by hydrolysing its bound GTP to GDP. GTP has a guanine attached to a ribose, which attaches to three sequential phosphate groups: O-P-O (-P-O etc) and the P has a single bond to both O- and and a double bond to O so that each P is bound to four Os.

The critical determinant of G-protein conformation is the y (third) phosphate of GTP, which interacts with a region called the P loop (phosphate-binding). In Ras, the y phosphate of GTP binds to a Lys residue in the P loop and to two critical residues, Thr^35 in switch I and Gly^60 in switch II, that hydrogen-bond with the oxygens of the y phosphate of GTP. These hydrogen bonds act like a pair of springs holding the
protein in its active conformation. When GTP is cleaved to GDP and Pi is released, these hydrogen bonds are lost; the protein relaxes into its inactive conformation, burying the sites that interact with other partners in its active state. Ala^146 hydrogen- bonds to the guanine oxygen, allowing GTP, but not ATP, to bind.

Question 53

What alternative name has been given to GAPs in the case of heterotrimeric G proteins?

Answer 53

The intrinsic GTPase activity of G proteins is increased up to 10 -fold by GTPase activator proteins (GAPs), also called, in the case of heterotrimeric G proteins, regulators of G protein signaling (RGSs)

Question 54

How do these these GAPs or RGSs contribute to the signalling? How exactly do they do this?

Answer 54

GAPs (and RGSs) thus determine how long the switch remains “on.” They contribute a critical Arg residue that reaches into the G-protein GTPase active site and assists in catalysis.

Question 55

How is the process of replacing bound GDP with GTP regulated?

Answer 55

The intrinsically slow process of replacing bound GDP with GTP, switching the protein on, is catalysed by guanosine nucleotide–exchange factors (GEFs) associated with the G protein

Question 56

Name three of these GEFs

Answer 56

Rh*
B-AR*
Sos
etc

Question 57

Because G proteins play crucial roles in so many signaling processes, it is not surprising that defects in G proteins lead to a variety of diseases. How does a mutation of Ras affect one huge disease?

Answer 57

In about 25% of all human cancers (and in a much higher proportion of certain types of cancer), there is a mutation in a Ras protein—typically in one of the critical residues around the GTP-binding site or in the P loop—that virtually eliminates its GTPase activity. Once activated by GTP binding, this Ras protein remains constitutively active, promoting cell division in cells that should not divide.

Question 58

How can mutations in other genes not regarding the g protein or signalling molecule affect G protein signal and incur cancer?

Answer 58

Mutations in NF1 that result in a non- functioning GAP leave Ras with only its intrinsic GTPase activity, which is very weak (has a very low turnover number); once activated by GTP binding, Ras stays active for an extended period, continuing to send the signal: divide.

Question 59

How can mutations in the g proteins themselves cause disease?

Answer 59

Defective heterotrimeric G proteins can also lead to disease. Mutations in the gene that encodes the a subunit of Gs (which mediates changes in [cAMP] in response to hormonal stimuli) may result in a Ga that is permanently active or permanently inactive.

Individuals with “inactivating” mutations in Ga are unresponsive to hormones (such as thyroid hormone) that act through cAMP. Mutation in the gene for the transducin a subunit (Ta), which is involved in visual signaling, leads to a type of night blindness, apparently due to defective interaction between the activated T􏰃 subunit and the phosphodies- terase of the rod outer segment (see Fig. 12–39). A sequence variation in the gene encoding the 􏰀 subunit of a heterotrimeric G protein is commonly found in indi- viduals with hypertension (high blood pressure), and this variant gene is suspected of involvement in obesity and atherosclerosis.

Question 60

Describe how a mutation leading to an overactive a subunit might look

Answer 60

“Activating” mutations generally occur in residues crucial to GTPase activity; they lead to a continuously elevated [cAMP], with significant downstream consequences, including undesirable cell proliferation. For example, such mutations are found in about 40% of pituitary tumors (adenomas).

Question 61

Describe how a mutation leading to an inactive a subunit might look

Answer 61

Individuals with “inactivating” mutations in Ga are unresponsive to hormones (such as thyroid hormone) that act through cAMP. Mutation in the gene for the transducin a subunit (Ta), which is involved in visual signaling, leads to a type of night blindness, apparently due to defective interaction between the activated Ta subunit and the phosphodiesterase of the rod outer segment.

A sequence variation in the gene encoding the a subunit of a heterotrimeric G protein is commonly found in individuals with hypertension (high blood pressure), and this variant gene is suspected of involvement in obesity and atherosclerosis.

Question 62

Name two toxins which target GPCRs

Answer 62

Cholera toxin

Pertussis toxin

Question 63

Where may Cholera toxin arise from?

Answer 63

Cholera toxin, secreted by Vibrio cholerae in the intestine of an infected person, is a heterodimeric protein.

Question 64

What is the mechanism of cholera toxin?

Answer 64

Subunit B recognises and binds to specific gangliosides on the surface of intestinal epithelial cells and provides a route for subunit A to enter these cells.

After entry, subunit A is broken into two pieces: the A1 fragment and the A2 fragment. A1 then associates with the ADP-ribosylation factor ARF6, a small G protein in host cells, through residues in its switch I and switch II regions—which are accessible only when ARF6 is in its active (GTP-bound) form.

This association with ARF6 activates A1, which catalyses the transfer of ADP-ribose from NAD+ to the critical Arg residue in the P loop of the a subunit of Gs. ADP-ribosylation blocks the GTPase activity of Gs and thereby renders Gs permanently active.

This results in continuous activation of the adenylyl cyclase of intestinal epithelial cells, chronically high [cAMP], and chronically active PKA. PKA phosphorylates the CFTR Cl- channel and a Na+-H+ exchanger in the intestinal epithelial cells.

Question 65

What is the outcome of cholera toxin?

Answer 65

The resultant efflux of NaCl triggers massive water loss through the intestine as cells respond to the ensuing osmotic imbalance. Severe dehydration and electrolyte loss are the major pathologies in cholera. These can be fatal in the absence of prompt rehydration therapy.

Question 66

What produces the pertussis toxin?

Answer 66

The pertussis toxin, is produced by Bordetella pertussis

Question 67

What is the mechanism of the pertussis toxin? What is the result?

Answer 67

The pertussis toxin, produced by Bordetella pertussis, catalyzes ADP-ribosylation of the a subunit of Gi, in this case preventing GDP-GTP exchange and blocking inhibition of adenylyl cyclase by Gi. The bacterium infects the respiratory tract, where it destroys the ciliated epithelial cells that normally sweep away mucus.

Without this ciliary action, vigorous coughing is needed to clear the tract; this is the gasping cough that gives the disease its name (and spreads the bacterium to others). How the defect in G-protein signalling kills ciliated epithelial cells is not yet clear.

Question 68

Epinephrine is just one of many hormones, growth factors, and other regulatory molecules that act by changing the intracellular [cAMP] and thus the activity of PKA. Give two examples of this involving transmembrane receptors

Answer 68

Glucagon binds to its receptors in the plasma membrane of adipocytes, activating (via a Gs protein) adenylyl cyclase. PKA, stimulated by the resulting rise in [cAMP], phosphorylates and activates two proteins critical to the mobilisation of the fatty acids of stored fats

Similarly, the peptide hormone ACTH (adrenocorticotropic hormone, also called corticotropin), produced by the anterior pituitary, binds to specific receptors in the adrenal cortex, activating adenylyl cyclase and raising the intracellular [cAMP]. PKA then phosphorylates and activates several of the enzymes required for the synthesis of cortisol and other steroid hormones.

Question 69

How can PKA affect transcription?

Answer 69

In many cell types, the catalytic subunit of PKA can also move into the nucleus, where it phosphorylates the cAMP response element binding protein (CREB), which alters the expression of specific genes regulated by cAMP.

Question 70

What effect does somatostatin have on PKA?

Answer 70

Some hormones act by inhibiting adenylyl cyclase, thus lowering [cAMP] and suppressing protein phosphorylation. For example, the binding of somatostatin to its receptor leads to activation of an inhibitory G protein, or Gi, structurally homologous to Gs, that inhibits adenylyl cyclase and lowers [cAMP]. Somatostatin therefore counterbalances the effects of glucagon.

Question 71

How else may glucagon be inhibited?

Answer 71

In adipose tissue, prostaglandin E1 (PGE1) inhibits adenylyl cyclase, thus lowering [cAMP] and slowing the mobilisation of lipid reserves triggered by epinephrine and glucagon.

Question 72

What other roles can prostaglandin E1 have?

Answer 72

In certain other tissues PGE1 stimulates cAMP synthesis: its receptors are coupled to adenylyl cyclase through a stimulatory G protein, Gs.

Question 73

How is epinephrine similar to PGE1 in this respect?

Answer 73

In tissues with a2-adrenergic receptors, epinephrine lowers [cAMP]; in this case, the receptors are coupled to adenylyl cyclase through an inhibitory G protein, Gi.

Question 74

In short, an extracellular signal such as epinephrine or PGE1 can have quite different effects on different tissues or cell types, depending on three factors. Name these

Answer 74

• The type of receptor in the tissue
• The type of G protein (Gs or Gi) with which the receptor is coupled
• The set of PKA target enzymes in the cells.

Question 75

What is another factor that explains how so many types of signals can be mediated by a single second messenger (cAMP)?

Answer 75

Another factor that explains how so many types of signals can be mediated by a single second messenger (cAMP) is the confinement of the signalling process to a specific region of the cell by adaptor proteins— noncatalytic proteins that hold together other protein molecules that function in concert.

Question 76

Describe one such adaptor protein that is involved in the B-adrenergic system

Answer 76

AKAPs (A kinase anchoring proteins) are multivalent adaptor proteins; one part binds to the R subunits of PKA and another to a specific structure in the cell, confining the PKA to the vicinity of that structure. For example, specific AKAPs bind PKA to microtubules, actin filaments, ion channels, mitochondria, or the nucleus.

Question 77

How may AKAPs change the function of a given signal

Answer 77

Different types of cells have different complements of AKAPs, so cAMP might stimulate phosphorylation of mitochondrial proteins in one cell and phosphorylation of actin filaments in another.

In some cases, an AKAP connects PKA with the enzyme that triggers PKA activation (adenylyl cyclase) or terminates PKA action (cAMP phosphodiesterase or phosphoprotein phosphatase)

Question 78

What characteristics of the PKA response are reflected in these variations of supramolecular complexes by AKAP?

Answer 78

The very close proximity of these activating and inactivating enzymes presumably achieves a highly localised, and very brief, response.

Question 79

As is now clear, to fully understand cellular signal- ing, researchers need tools precise enough to detect and study the spatiotemporal aspects of signaling processes at the subcellular level and in real time. Describe a technique, aside from co-immunoprecipitation which would allow you to study these protein interactions

Answer 79

Changes in the state of association of two proteins (such as the R and C sub- units of PKA) can be seen by measuring the nonradiative transfer of energy between fluorescent probes attached to each protein, a technique called fluorescence resonance energy transfer (FRET)

Question 80

Comment on the temporal and spatial resolution of flourescent probes and microscopy

Answer 80

Flourescent probes can be designed to give an essentially instantaneous report (within nanoseconds) on the changes in intra- cellular concentration of a second messenger or in the activity of a protein kinase. Furthermore, fluorescence microscopy has sufficient resolution to reveal where in the cell such changes are occurring.

Question 81

Describe how GFP works endogenously

Answer 81

When excited by absorption of a photon of light, GFP emits a photon (that is, it fluoresces) in the green region of the spectrum. The light-absorbing/ emitting center of GFP (its chromophore) comprises an oxidized form of the tripeptide –Ser^65 –Tyr^66 –Gly^67 –. Oxidation of the tripeptide is catalyzed by the GFP protein itself

Question 82

Why is GFP useful?

Answer 82

Oxidation of the tripeptide is catalysed by the GFP protein itself, so it is possible to clone the protein into virtually any cell, where it can serve as a fluorescent marker for any protein to which it is fused. GFP and its variants are compact structures that retain their ability to fold into their native B-barrel conformation even when fused with another protein.

Question 83

Describe variants of GFP

Answer 83

Variants of GFP, with different fluorescence spectra, are produced by genetic engineering. For example, in the yellow fluorescent protein (YFP), Ala206 in GFP is replaced by a Lys residue, changing the wavelength of light absorption and fluorescence. Other variants of GFP fluoresce blue (BFP) or cyan (CFP) light, and a related protein (mRFP1) fluoresces red light.

Question 84

An excited fluorescent molecule such as GFP or YFP can dispose of the energy from the absorbed photon in either of two ways. What are these two ways?

Answer 84

(1) by fluorescence, emitting a photon of slightly longer wavelength (lower energy) than the exciting light

(2) by nonradiative fluorescence resonance energy transfer (FRET), in which the energy of the excited molecule (the donor) passes directly to a nearby molecule (the acceptor) without emission of a photon, exciting the acceptor

Question 85

What happens with the acceptor when it disposes of the energy by flourescence?

Answer 85

The acceptor can now decay to its ground state by fluorescence; the emitted photon has a longer wavelength (lower energy) than both the original exciting light and the fluorescence emission of the donor.

Question 86

When is this second mode of decay (FRET) possible?

Answer 86

This second mode of decay (FRET) is possible only when donor and acceptor are close to each other (within 1 to 50 Å; Angstrom: 1/10 of a nanometer); the efficiency of FRET is inversely proportional to the sixth power of the distance between donor and acceptor.

Question 87

What functional implications does this condition of FRET have?

Answer 87

Thus very small changes in the distance between donor and acceptor register as very large changes in FRET, measured as the fluorescence of the acceptor molecule when the donor is excited. With sufficiently sensitive light detectors, this fluorescence signal can be located to specific regions of a single, living cell.

Question 88

How has FRET been used to image a physiological process in cells? (most famous)

Answer 88

FRET has been used to measure [cAMP] in living cells. The gene for GFP is fused with that for the regulatory subunit (R) of cAMP-dependent protein kinase (PKA), and the gene for BFP is fused with that for the catalytic subunit (C). When these two hybrid proteins are expressed in a cell, BFP (donor; excitation at 380 nm, emission at 460 nm) and GFP (acceptor; excitation at 475 nm, emission at 545 nm) in the inactive PKA (R2C2 tetramer) are close enough to undergo FRET. Wherever in the cell [cAMP] increases, the R2C2 complex dissociates into R2 and 2C and the FRET signal is lost, because donor and acceptor are now too far apart for efficient FRET.

Question 89

How can FRET, being used to analyse [cAMP], be analysed and inferences be made?

Answer 89

Viewed in the fluorescence microscope, the region of higher [cAMP] has a minimal GFP signal and higher BFP signal. Measuring the ratio of emission at 460 nm and 545 nm gives a sensitive measure of the change in [cAMP]. By determining this ratio for all regions of the cell, the investigator can generate a false colour image of the cell in which the ratio, or relative [cAMP], is represented by the intensity of the colour. Images recorded at timed intervals reveal changes in [cAMP] over time.

Question 90

How does FRET techniques relate to GPCRs?

Answer 90

A variation of this technology has been used to measure the activity of PKA in a living cell. Researchers create a phosphorylation target for PKA by producing a hybrid protein containing four elements: YFP (acceptor); a short peptide with a Ser residue surrounded by the consensus sequence for PKA; a P–Ser-binding domain (called 14-3-3); and CFP (donor). When the Ser residue is not phosphorylated, 14-3-3 has no affinity for the Ser residue and the hybrid protein exists in an extended form, with the donor and acceptor too far apart to generate a FRET signal.

Wherever PKA is active in the cell, it phosphorylates the Ser residue of the hybrid protein, and 14-3-3 binds to the P–Ser. In doing so, it draws YFP and CFP together and a FRET signal is detected with the fluorescence microscope, revealing the presence of active PKA.

Question 91

Describe in more detail how you would use flourescence to measure cAMP with FRET

Answer 91

Gene fusion creates hybrid proteins that exhibit FRET when the PKA regulatory (R) and catalytic (C) subunits are associated (low [cAMP]). When [cAMP] rises, the subunits dissociate and FRET ceases due to the departure of the catalytic unit. The ratio of emission at 460 nm (dissociated) and 545 nm (complexed) thus offers a sensitive measure of [cAMP].

Question 92

Describe again how you can measure the activity of PKA with FRET

Answer 92

An engineered protein links YFP and CFP via a peptide that contains a Ser residue surrounded by the consensus sequence for phosphorylation by PKA, and the 14-3-3 P –Ser-binding domain. Active PKA phosphorylates the Ser residue, which docks with the 14-3-3 binding domain, bringing the fluorescence proteins close enough to allow FRET to occur, revealing the presence of active PKA.