Module 10 Worksheet

Why do you suppose that phosphorylation/dephosphorylation, as opposed to allosteric binding of small molecules, for example, has evolved to play such a prominent role in switching proteins off and on in signaling pathways?

-verstile -quick -easily reversed allosteric -very specific -needs coded -may or may not be easily reveresed -slower

Summarize the steps in the cycling of GTPase switch proteins from active to inactive states.

1. GTPase switch proteins are active when they are bound to GTP. 2. GTPase-activating proteins (GAPs) and regulators o f G protein signaling (RGSs) accelerate the hydrolysis of the bound GTP, converting the active GTPase switch protein to the inactive version. 3. The inactive GTPase switch protein is bound to GDP. 4. Guanine nucleotide exchange factors (GEFs) facilitate the reactivation of the GTPase switch protein, which dissociates from GDP and binds to GTP, starting the cycle over.

What is the purpose of A kinase-associated proteins (AKAPs)? Describe how AKAPs work in heart muscle cells.

AKAPs localize PKA activity to certain regions of cells to increase the speed of the signaling response. AKAP15 protein localizes PKA next to Ca2+ channels in heart muscle cells, which reduces the time that otherwise would be required for diffusion of PKA catalytic subunits from their sites of activation to the Ca2+ channel substrates. A different AKAP in heart muscle anchors both PKA and cAMP phosphodiesterase (PDE) to the outer nuclear membrane. Localization of PKA at the nuclear membrane facilitates rapid entry of some of the activated PKA catalytic subunits into the nucleus, where they phosphorylate transcription factors. The proximity of the PDE provides for tight localized control of PKA activity through a negative feedback mechanism in which PKA phosphorylation of the PDE accelerates destruction of the cAMP thereby reactivating PKA inhibition.

A hormone binds a receptor and activates the cAMP signal transduction pathway ultimately leading to the cell response. Which of the following is NOT a likely mechanism for downregulating cAMP transduction? A) The hormone dissociating from the receptor. B) The GTP on the alpha subunit of the G protein is hydrolyzed to GDP. C) Activate phosphodiesterase. D) Activate adenylyl cyclase. E) Protein phosphatases remove phosphates from proteins initially activated by protein kinase A

Activate adenylyl cyclase.

Describe the steps in the synthesis of 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) from phosphatidylinositol (PI). What do these second messengers do?

After a ligand activates a trimeric G protein, the alpha subunit bind to and activates phospholipase C which cleaves PI(4,5)P2 into IP3 and DAG. IP3 controls the release of Ca2+ into the cytosol, that is, when it binds to the ligand gated ion channel of the endoplasmic reticulum, Ca2+ is released. DAG activates protein kinase C which phosphorylates and activates several other proteins.

Describe the differences between an agonist and an antagonist. If a particular antagonist for an epinephrine-receptor protein is under consideration as a new drug, what values would you use to measure how tightly the drug binds to the target protein compared to the tightness of epinephrine binding?

Agonists mimic ligands and bind to a receptor to induce a certain response while antagonists are the opposite and bind to a receptor and don't induce a response. To measure how tightly the drug binds to the target protein, we would want to look at Kd, the dissociation constant which measures how tightly a ligand binds to its receptor. If the Kd of the drug is less than that of epinephrine, then that would mean the drug binds more tightly and vice versa. Additionally, you we could also utilize a competetive binding assay.

Explain how FRET could be used to monitor the association of Gαs and adenylyl cyclase following activation of the epinephrine receptor.

As in Figure 15-18, the Gas protein could be expressed as a CFP-fusion protein and adenylyl cyclase, instead of Gby could be expressed as a YFP-fusion protein. In this case, association of Gas with adenylyl cyclase would yield an increase in energy transfer and fluorescence at 527 nm when excited with 440 nm light.

How do seven-transmembrane domain G protein-coupled receptors transmit a signal across the plasma membrane? In your answer, include the conformational changes that occur in the receptor in response to ligand binding.

Binding of the extracellular signal or, in the case of rhodopsin, absorption of a photon by retinal, causes a conformational change in the orientation of the transmembrane helices that changes the conformation of the C3 loop and C4 segment (and in some cases the C2 loop) on the cytosolic side of the receptor. This increases the affinity of the receptor cytosolic region for binding and activating a G protein.

Both rhodopsin in vision and muscarinic acetylcholine receptor system in cardiac muscle are coupled to ion channels via G proteins. Describe the similarities and differences between these two systems.

Both rhodopsin and muscarinic acetylcholine receptor are similar in that they are GPCRs. Furthermore, the binding of the ligand (or absorption of photon in rhodopsin) to the Gαt triggers the exchange of GTP for GDP on the Gαt subunit. After this, both GPCRs dissciate into their Gαt and Gβγ subunits. Furthermore when both of these receptor systems are activated, both membrane depolarize. The muscarinic acetylcholine receptor system in cardiac muscle is different from rhodopsin in that the binding of acetylcholine leads to the opening of K+ ion channels whereas in rhodopsin it is the absorption of a photon of light that activates the receptor and the ion channels involved are Na+ and Ca2+ Also, with the muscarinic acetylcholine receptor system the Gβγ protein, rather than Gαi like in rhodopsin, opens the ion channels.

In paracrine signaling

Cells release a signal that affects neighboring cells.

Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) are second messenger molecules derived from the cleavage of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] by activated phospholipase C. Describe the role of IP3 in causing a rise in cytosolic Ca2+ concentration. How do cells restore resting levels of cytosolic Ca2+? What is the principal function of DAG?

Cleavage of PIP2 by phospholipase C generates IP3 and DAG. IP3 opens IP3-gated Ca2+ channels in the endoplasmic reticulum (ER) membrane, resulting in release of Ca2+ from the ER. When ER stores of Ca2+ are depleted, the IP3-gated Ca 2+ channels bind to and open store-operated Ca 2+ channels in the plasma membrane, allowing an influx of Ca2+ To restore resting levels of cytosolic Ca2+, Ca2+-ATPase pumps located in the ER membrane and in the plasma membrane pump cytosolic Ca2+ back into the ER lumen and out of the cell. The principal function of DAG is to activate protein kinase C, which then phosphorylates specific target proteins.

Explain the differences between endocrine, paracrine, and autocrine signaling.

Endocrine signaling: only one organ synthesizes the signaling molecules and then these molecules act on cells that are farther away in the body by traveling via the blood or extrecellular fluid. Paracrine signaling: the signaling molecules only reach cells that are close by. Autocrine signaling: the signaling molecules affect the cell that released them

Epinephrine binds to both β-adrenergic and α-adrenergic receptors. Describe the opposite actions on the effector protein, adenylyl cyclase, elicited by the binding of epinephrine to these two types of receptors. Describe the effect of adding an agonist or antagonist to a β-adrenergic receptor on the activity of adenylyl cyclase.

Epinephrine binding to the b-adrenergic receptor causes an activation of adenylyl cyclase through the activation of Gas, a stimulatory G protein. In contrast, epinephrine binding to the a-adrenergic receptor causes an inhibition of adenylyl cyclase through the activation of GaI, an inhibitory G protein. An agonist acts like the normal hormone, which in this case would be epinephrine. Thus, agonist binding to a b-adrenergic receptor would result in activation of adenylyl cyclase. In contrast, an antagonist binds to the receptor but does not activate the receptor. Thus, antagonist binding to a b-adrenergic receptor would have no effect on adenylyl cyclase activity. In fact, it would reduce a normal epinephrine stimulated response because it would prevent epinephrine from binding to the receptor.

What common features are shared by most cell signaling systems?

Extracellular signals are made by signaling cells. Receptor proteins are present in target cells. Binding of extracellular signaling molecules to cell-surface receptors triggers a conformational change in the receptor, which in turn leads to intracellular signal-transduction pathways that ultimately modulate cellular metabolism, function, or gene expression. Intracellular signal transduction pathways are evolutionarily highly conserved.

Which of the following is a correct association? kinase activity and the addition of a tyrosine phosphodiesterase activity and the removal of phosphate groups GTPase activity and hydrolysis of GTP to GDP phosphorylase activity and the catabolism of glucose adenylyl cyclase activity and the conversion of cAMP to AMP

GTPase activity and hydrolysis of GTP to GDP

Growth hormone is secreted from the pituitary, which is located at the base of the brain and acts through growth hormone receptors located on the liver. Is this an example of endocrine, paracrine, or autocrine signaling? Why?

Growth hormone is an example of endocrine signaling because the growth hormone is synthesized in the pituitary, located at the base of the brain, and travels to the liver via the blood.

Which of the following order of events is most accurate for activation of G-protein coupled receptor (GPCR) signaling pathways?

Hormone binds GPCR, GPCR binds with G protein, G protein loses GDP and gains GTP, G-protein alpha subunit activates intracellular proteins.

Most of the short-term physiological responses of cells to cAMP are mediated by activation of PKA. Another common second messenger is cGMP. What are the targets of cGMP in rod and smooth muscle cells?

In rod cells, cGMP opens cation channels, whereas the primary activity of cGMP in smooth muscle cells, like cAMP, is to activate a kinase.

Which of the following is the best explanation for the fact that most transduction pathways have multiple steps? Most of the steps were already in place because they are steps in other pathways. Multiple steps in a pathway require the least amount of ATP. Multiple steps provide for greater possible amplification of a signal. Each individual step can remove excess phosphate groups from the cytoplasm. Each step can be activated by several G proteins simultaneously.

Multiple steps provide for greater possible amplification of a signal.

The activity of β-adrenergic receptors may be regulated by:

Phosphorylation by β-adrenergic receptor kinase (BARK). Homologous desensitization Heterologous desensitization Intracellular sequestration of the receptor

In Chapter 3, the Kd of calmodulin EF hands for binding Ca2+ is given as 10−6 M. Many proteins have much higher affinities for their respective ligands. Why is the specific affinity of calmodulin important for Ca2+ signaling processes such as that initiated by production of IP3?

Prior to stimulation of the IP3-DAG signaling pathway, Ca2+-ATPases establish a resting level of Ca2+ in the cytosol of <10-7M, at which level few calmodulin sites have Ca2+ bound and the calmodulin is inactive. IP3 stimulates a rise in cytosolic Ca2+ concentration to >10-5M, at which level most calmodulin sites have Ca2+ bound and calmodulin is active. The Ca2+ binding affinity of calmodulin is exquisitely tuned to bind and release Ca2+ in response to the physiological changes of Ca2+ concentration in the cytoplasm used for signaling.

Diacylglycerol (DAG) activates : protein kinase A protein kinase C calmodulin Protein kinase G phospholipase C

Protein Kinase C

Continuous exposure of a Gαs protein-coupled receptor to its ligand leads to a phenomenon known as desensitization. Describe several molecular mechanisms for receptor desensitization. How can a receptor be reset to its original sensitized state? What effect would a mutant receptor lacking serine or threonine phosphorylation sites have on a cell?

Receptor desensitization can involve phosphorylation of the receptor itself. The increase in cAMP levels as a result of ligand binding to the receptor leads to an activation of protein kinase A. Protein kinase A can phosphorylate target proteins as well as cytosolic serine and threonine residues in the receptor itself. Phosphorylated receptor can bind ligand but is reduced in its ability to activate adenylyl cyclase. Thus, the receptor is desensitized to the effect of ligand bind- ing. Phosphorylated receptors are resensitized by the removal of phosphates by phosphatases. A mutant receptor that lacked serine or threonine phosphorylation sites could be resistant to desensitization by phosphorylation and thus would continuously activate adenylyl cyclase in the presence of ligand.

Describe the various molecular mechanisms for receptor desensitization. How can a receptor be reset to its original sensitized state?

Receptors can be desensitized by phosphorylation of the receptor itself. The phosphorylated receptor can bind the ligand but cannot activate adenylyl cyclase. Therefore, it cannot cause an increase in cAMP, and thus no activation of protein kinase A or target proteins. Thus, the receptor pathway is desensitized to the effect of ligand binding. Phosphorylated receptors are reset to the original sensitized state when phosphatase enzymes remove phosphate from the receptor. 3. Describe the various molecular mechanisms for receptor desensitization. How can a receptor be reset to its original sensitized state? Homologous desensitization of a receptor occurs when a receptor is under constant influence of an agonist. After so long, the receptor will lighten the response of the cascade events it triggers (basically decreasing the signal) when the agonist is at a high concentration. Heterologous desensitization is similar in which it no longer can react to an agonist. After so many bindings of an agonist, the receptor no longer will activate a pathway. There are more specific mechanisms to talk about such as receptor uncoupling and endocytosis. Receptor uncoupling uses beta androgenic receptor kinases to add a phosphate to the receptor. Beta arrestin has an increased affinity for receptors that are phosphorylated and active. The receptor is uncoupled resulting in a desensitized receptor. GCPR endocytosis can be similarly phosphorylated, but will be taken into the cytoplasm and degraded by lysosomes or put back in the plasma membrane later.

Which of the following steps amplify the epinephrine signal response in cells: receptor activation of G protein, G protein activation of adenylyl cyclase, cAMP activation of PKA, or PKA phosphorylation of glycogen phosphorylase kinase (GPK)? Which change will have a greater effect on signal amplification: an increase in the number of epinephrine receptors or an increase in the number of Gαs proteins?

Steps at which a single active component activates multiple targets amplify the signal. Each active receptor activates multiple Gas proteins, and each active PKA initiates a short kinase cascade by phosphorylating multiple GPKs, each of which phosphorylates multiple glycogen phosphorylase enzymes to continue the signal amplification. In contrast, each active Gas protein activates only one AC, and each cAMP participates in the activation of only one PKA, neither of which directly amplifies the signal response. An increase in the number of receptors would have a greater effect on amplification because proportionally more Gas proteins would be activated for the same level of epinephrine, whereas if only the number of Gas proteins were increased, the signal response would still depend on the number of receptors activated.

Following its initial activation, how does a G protein become inactivated? -It spontaneously loses activity within a few seconds. -The G-protein α subunit containing a GTP eventually reassociates with the G-protein βγ dimer. -The G-protein α subunit GTP is hydrolyzed to GDP allowing reassociation of the G-protein α subunit and βγ dimer. -The receptor becomes internalized. -The G-protein α subunit is digested and recycled.

The G-protein α subunit GTP is hydrolyzed to GDP allowing reassociation of the G-protein α subunit and βγ dimer.

In liver and muscle, epinephrine stimulation of the cAMP pathway activates glycogen breakdown and inhibits glycogen synthesis, whereas in adipose tissue, epinephrine activates hydrolysis of triglycerides, and in other cells, it causes a diversity of other responses. What step in the cAMP signaling pathways in these cells specifies the cell response?

The epinephrine-cAMP signaling pathway—from binding of epinephrine to the receptor to activation of PKA—is essentially the same in all the cells. The downstream biochemical pathway activated is specified by the substrate(s) phosphorylated by PKA.

A ligand binds two different receptors with a Kd value of 10−7 M for receptor 1 and a Kd value of 10−9 M for receptor 2. For which receptor does the ligand show the greater affinity? Calculate the fraction of receptors that have a bound ligand ([RL]/RT) in the case of receptor 1 and receptor 2 if the concentration of free ligand is 10−8 M.

The ligand-receptor complex that shows the lower Kd value has the higher affinity. Because the Kd for receptor 2 (10−9M) is lower than that for receptor 1 (10−7M), the ligand shows greater affinity for receptor 2 than for receptor 1. To calculate the fraction of receptors with bound ligand, [RL]/RT, use Equation 15-2 [RL]/R T = 1/(1 + Kd/[L]). For receptor 1, the Kd is 10−7M and the concentration of free ligand [L] is 10−8M. Thus, the [RL]/RT for receptor 1 is 0.091, that is, only 9% of the receptors have bound ligand at a free ligand concentration of 10−8 M. In contrast, the [RL]/RT for receptor 2 is 0.91; 91% of the receptors have bound ligand.

Review the steps for ligand-induced activation of effector protein mediated by trimeric G proteins. Suppose you have isolated a mutant Gα that has increased GTPase activity, what effect would this mutation have on the G protein and the effector protein?

The steps for ligand-induced activation of effector protein mediated by trimeric G proteins are as follows: a ligand bind the G protein-coupled receptor, causing a conformational change that allows the receptor to bind to the Gα subunit of the G protein. This binding causes the release of GDP and then GTP takes its place at the Gα subunit. Finally, the Gα subunit binds less tightly to the Gβγ subunit and is then able to interact with an effector protein. Increased GTPase activity means that there is a higher rate of hydrolyzation of GTP to GDP and thus a deactivation of the G protein and, consequently, lower rates of activation for the effector protein.

Review the steps for ligand-induced activation of effector protein mediated by trimeric G proteins. Suppose you have isolated a mutant Gα that has increased GTPase activity, what effect would this mutation have on the G protein and the effector protein?

The steps for ligand-induced activation of effector protein mediated by trimeric G proteins are: Ligand binds to the GPCR, causing a conformational change that allows the GPCR to bind to the Gα subunit of the G protein. GPCR binding to Gα causes the release of GDP and then binding of GTP to the Gα subunit. The Gα subunit binds less tightly to the Gβγ subunit and is then the G protein is able to interact with the effector protein. Having a mutant Gα with increased GTPase activity would lead to a higher rate of GTP hydrolysis and thus a deactivation of the G protein and, consequently, lower rates of activation for the effector protein.

To understand how a signaling pathway works, it is often useful to isolate the cell-surface receptor and to measure the activity of downstream effector proteins under different conditions. How could you use affinity chromatography to isolate a cell-surface receptor? With what technique could you measure the amount of activated G protein (the GTP-bound form) in ligand-stimulated cells? Describe the approach you would take.

To purify a receptor by affinity chromatography, a ligand for the receptor is chemically linked to beads used to form a column. A detergent-solubilized cell membrane extract containing the receptor is then passed through the column. The receptor will bind to the ligand attached to the beads and other proteins will wash out. The receptor can then be eluted from the column with an excess of ligand. Analyzing its molecular weight by SDS-PAGE may be sufficient to identify the recovered receptor or provide protein for sequencing. A similar column chromatography approach or an alternative affinity approach known as a pull-down assay can be used to isolate activated G protein. To isolate activated G protein with a pull-down assay, the adenylyl cyclase (AC) effector enzyme to which only the active form of G protein binds is linked to beads either directly by chemical linkage or indirectly through an anti-AC antibody bound to protein A beads. Mixing the AC-coupled beads with a cell extract and then pelleting the beads by centrifugation pulls down only active G protein because inactive G protein (the GDP-bound form) fails to interact with the effector enzyme. The amount of activated G protein pulled down can be quantified under different signaling conditions.

Describe how acetylcholine mediates arterial smooth muscle relaxation and vasodialation.

Vascular endothelial cells have a GPCR that has the ability to bind acetylcholine. If this happens, phospholipase C is activated and then an increased amount of Ca2+ appears in the cytosol. Then Ca2+ binds to calmodulin and this unit promotes the production of NO. NO is essentially the molecule that promotes smooth muscle relaxation and vasodilation. The NO stimulates the guanylyl cyclase to increase cyclic guanosine monophosphate (cGMP) in the vascular smooth muscle to cause relaxation.

6. How does the body respond to decreases in blood glucose levels below about 5 mM?

Your body knows that it needs sugar. On the exterior of the liver, the liver secretes epinephrine which stimulates the release of glucose. Epinephrine will bind to Beta-adrenergic receptor protein on the outside and will never cross the plasma membrane due to its hydrophilic properties. G proteins on the inside of the liver are activated by the attachment of epinephrine to the Beta-adrenergic receptor. The G proteins (3 subunits total) will lose one of the subunits. The subunit that disassociated from the other two had a GDP molecule that is replaced by GTP. It will move until it binds to Adenylyl cyclase in the membrane which then catalyzes the formation of cAMP from ATP. cAMP will travel in the cytoplasm until it reaches Protein Kinase A. Protien Kinase A phosphorylates another enzyme called phosphorylase and activates it. Phosphorylase then converts glycogen to glucose-6-phosphate. Then, glucose-6 phosphate is then converted to glucose.

Calmodulin

a calcium-binding protein that regulates such cell activities as muscle contractions

Which of the following events occur(s) during the hormone-stimulated conversion of glycogen to glucose-1-phosphate?

activation of beta-adrenergic receptor upon binding to epinephrine. Activation of PKA upon binding to cAMP inhibition of glycogen synthase activation of glycogen phosphorylase

phospholipase C

an enzyme that catalyzes the splitting of a specific type of phospholipid into IP3 and DAG (both of which act as second messenger molecules).

A major group of G protein-coupled receptors contains seven transmembrane α helices. The amino end of the protein lies at the exterior of the plasma membrane. Loops of amino acids connect the helices either at the exterior face or on the cytosol face of the membrane. The loop on the cytosol side between helices 5 and 6 is usually substantially longer than the others. The coupled G protein most likely interacts with this receptor where?

at the carboxyl terminal end, the loop between H5 and H6 and other cytosol-facing loops.

The KD or dissociation constant between a receptor and its ligand is a measure of the binding affinity the receptor has for the hormone. the hormone concentration where half the receptors are bound to hormone. the hormone concentration where all of the receptors are bound to hormone. both a measure of the binding affinity the receptor has for the hormone and the hormone concentration where half the receptors are bound to hormone. both a measure of the binding affinity the receptor has for the hormone and the hormone concentration where all the receptors are bound to hormone.

both a measure of the binding affinity the receptor has for the hormone and the hormone concentration where half the receptors are bound to hormone

Phosphodiesterase activity is involved in the regulation of signal transduction because it

converts cGMP to GMP

Protein kinase A (PKA) is activated by

cyclic AMP, allosterically

Elevated epinephrine levels do not normally stimulate: A) fatty acid mobilization in adipose tissue. B) glycogen breakdown in liver. C) glycogen breakdown in muscle. D) glycogen synthesis in liver. E) glycolysis in muscle.

glycogen synthesis in liver

Lipid-soluble signaling molecules, such as testosterone, can cross the membranes of all cells but they affect and produce a response only in certain target cells. This may be because

intracellular receptors that recognize and bind to testosterone are present only in target cells.

What type of receptor does IP3 bind?

ligand-gated Ca2+ channel

The cholera toxin causes profuse diarrhea because it

modifies a G protein involved in regulating salt and water secretion.

Protein kinase C

part of second messenger system that is activated by diacylglycerol and results in the activation of proteins in the cell

Adenylyl cyclase is to cAMP as ________ is to IP3. protein kinase A protein kinase C calmodulin Protein kinase G phospholipase C

phospholipase C

Which of the following is LEAST likely involved in calcium signaling? A) IP3 B) calmodulin C) protein kinase A D) protein kinase B E) phospholipase C

protein kinase A

protein kinase A

responsible for the intracellualr effects of the G-protein/adenylate cyclase second messenger system . Glucagon, PTH, and TSH all use this method.

protein kinase G

this protein is cGMP dependent to phospohrylate proteins. In smooth muscle cells it drives a decrease in calcium.

The G-protein involved in visual signal transduction is

transducin