Biochem Chpt 12.2 GPCRs and second messengers Flashcards
What does the name GPCR tell regarding their behaviour?
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.
What three essential components define signal transduction through GPCRs?
- 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.
What happens when the G protein is stimulated?
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
How many GCPRs are encoded in the human genome?
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.
What is the clinical relevance of GCPRs?
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.
To what extent have these receptors been identified?
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.
What is the function of epinephrine?
Epinephrine sounds the alarm when some threat requires the organism to mobilise its energy-generating machinery; it signals the need to fight or flee.
What binds to what for epinephrine to function?
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.
What are meant by agonists and antagonists?
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
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?
muscle, liver, and adipose tissue
These receptors mediate changes in fuel metabolism, including the increased breakdown of glycogen and fat.
Why talk generally about the B-adrenergic receptors?
Adrenergic receptors of the B1 and B2 subtypes act through the same mechanism, so in our discussion, “B-adrenergic” applies to both types.
Describe the B-adrenergic receptor
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.
What name describes all G proteins which are involved in GPCRs?
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.
Broadly, how does epinephrine affect the B-Adregenergic receptors?
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.
Describe in regards to the individual subunits how this conformational effect of epinephrine on the B-adrenergic receptors take place
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
What is adenylyl cyclase?
Adenylyl cyclase is an integral protein of the plasma membrane, with its active site on the cytoplasmic face.
What does this association of adenylyl cyclase and Gsa incur?
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.
How does Gsa binding of adrenylyl cyclase relate to its own activity?
The stimulation by Gsa is self-limiting; Gsa has intrinsic GTPase activity that inactivates Gsa by converting its bound GTP to GDP
What happens to Gsa and adrenyl cyclase once it is inactive?
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.
What aspect of this function is reflective of a general role of G proteins?
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
How does epinephrine exert its downstream effects following this cascade?
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.
Describe the structure of inactive PKA
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).
How does this molecular cascade therefore affect PKA?
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.
How does PKA compare to other protein kinases? (2)
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.
What is the consequence of this activation of PKA?
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.
What has the comparison of various protein substrates of PKA achieved?
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.
What has the comparison of various protein substrates of PKA achieved?
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.
As in many signaling pathways, signal transduction by adenylyl cyclase entails several steps that amplify the original hormone signal. Describe these steps
- 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)
- By activating one molecule of adenylyl cyclase, each active Gsa molecule stimulates the catalytic synthesis of many molecules of cAMP. (200 molecules)
- 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)
- 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.
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
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.
What happens when the concentration of epinephrin in the blood drops below the Kd?
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.
Describe another intrinsic means of ending the B-adrenergic response
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.
What does the rate of inactivation of Gs depend on?
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
How are GAP levels determined?
GAPs can themselves be regulated by other factors, providing a fine-tuning of the response to B-adrenergic stimulation.
How may the response be terminated lower in the cascade? (2)
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).
How do the number of phosphoprotein phosphatases encoded by the genome compare to the number encoding protein kinases?
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.
What impact do these phosphoprotein phosphatases have in this cascade?
When [cAMP] drops and PKA returns to its inactive form, the balance between phosphorylation and dephosphorylation is tipped toward dephosphorylation by these phosphatases
Thus, briefly give 7 stages in B-adrenergic signalling
- Epinephrine binds to its
specific receptor. - Hormone-receptor complex causes the GDP bound to Gsa to be replaced by GTP, activating
Gsa. - Activated Gsa separates from GsBy, moves to adenylyl cyclase, and activates it. Many Gsa subunits may be activated by one occupied receptor.
- Adenylyl cyclase catalyses the formation of cAMP.
- cAMP activates PKA.
- Phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine.
- cAMP is degraded, reversing the activation of PKA.
