Biosignaling - Marquette University · Gated Ion Channels The NICOTINIC ACETYLCHOLINE RECEPTOR is...

Biosignaling

Objectives: I. Discuss the reasons for biosignaling and the information transduction systems. II. Discuss why the signals must be specific and sensitive.

A. Discuss factors that contribute to the specificity of the signal. B. Discuss factors that contribute to the sensitivity of the signal.

III. Be aware of the six (6) different receptor types / transduction systems. A. Gated Ion Channels. B. Trimeric G Protein Couples Receptors. C. Receptor Tyrosine Kinases. D. Receptor Guanylyl Cyclases. E. Nuclear Receptors. F. Adhesion Receptors

IV. Describe Gated Ion Channels as transduction systems. A. Describe the acetylcholine receptor as an example of a ligand gated channel.

1. Its structure. 2. Its function as a ligand gated channel and as part of an information transduction system.

B. Describe the functions of signal gated, voltage gated, and mechanosensitive channels as transduction systems.

V. Describe the general structures and functions of the trimeric G proteins. A. Structure B. Interaction with the cell membrane. C. Interaction with its receptor protein. D. Enzyme activity. E. Guanosine Nucleotide Exchange Factors. F. GTPase Activator Proteins.

VI. Transduction systems coupled to the trimeric G proteins. A. Describe the component parts of the transduction system coupled to the trimeric Gs protein. B. Describe the sequence the events involved in signal transduction coupled to the trimeric Gs

protein. C. What is the final cellular effect / response of the Gs system? D. Describe the component parts of the transduction system coupled to the trimeric Gi protein. E. Describe the sequence the events involved in signal transduction coupled to the trimeric Gi

protein. F. What is the final cellular effect / response of the Gi system? G. Describe the component parts of the transduction system coupled to the trimeric Gq protein. H. Describe the sequence the events involved in signal transduction coupled to the trimeric Gq

protein. I. What is(are) the final cellular effect(s) / response(s) of the Gq system? J. Function of Adaptor Proteins in trimeric G systems & transduction systems in general

1. AKAP79 2. AKAP250

VII. Tyrosine Kinase Receptors A. Describe the general structures and functions of the tyrosine kinase receptors.

©Kevin R. Siebenlist, 20191

1. What must happen to completely activate the kinase activity of these receptors? 2. What other enzyme(s) may be activated by / may be associated with these receptors?

B. Describe the general structure of the insulin receptor and how it is different from the general tyrosine kinase receptors. 1. Discuss the general effects brought about on the organism by insulin activating the Insulin

receptor. 2. Describe the general structures and functions of the small monomeric G proteins, such as

Ras. 3. Briefly, sequence the events involved in signal transduction coupled to the insulin receptor.

a) The pathway leading to increased protein synthesis. b) The pathway leading to increased glucose utilization.

4. Crosstalk between insulin receptor and the VIII.Receptor Guanylyl Cyclases

A. Structure. B. Enzymes Activity. C. Second Messenger. D. Downstream Effects.

1. Kinase activated. 2. Systemic Effects.

IX. Discuss how signal transduction systems gone awry can lead to abnormal cell growth. A. What type(s) of change(s) to the signal molecule can lead to abnormal cell growth? B. What type(s) of change(s) to the receptor can lead to abnormal cell growth? C. What type(s) of change(s) to the the G proteins can lead to abnormal cell growth? D. What type(s) of change(s) to the protein kinases can lead to abnormal cell growth?

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Background

Cells within complex organisms must be able to respond to a variety of internal and external stimuli. Signals to which cells respond include:

• Antigens • Cell Surface Glycoproteins • Developmental Signals • Extracellular Matrix Components • Growth factors • Hormones • Light • Mechanical Touch • Neurotransmitters • Odorants • Pheromones • Tastants

The response to these signals must be Specific and Sensitive


Two factors account for the Specificity of signal transduction in higher animals: 1. The signal molecule has a precise molecular complementarity with the cellular receptor protein. The

binding is mediated by weak intermolecular interactions similar to substrate binding to enzymes. 2. Receptor proteins for a particular signal molecule are present only on those cell types that need to

respond to the signal; e.g., hepatocytes do not contain the receptor for the neurotransmitter acetylcholine because they do not need to respond to this signal.

Three factors account for the Sensitivity of signal transduction: 1. High affinity of receptors for the signal molecules. The dissociation constant, Kd, of the receptor for

the signal molecule is often 10–10 M or smaller; i.e., the receptors are half saturated when the signal is present at pico molar concentrations or lower.

2. Cooperativity in the ligand-receptor interaction; binding of the first signal molecule makes binding of subsequent molecules easier. Cooperativity results in large changes in receptor activation with small changes in ligand concentration.

3. Amplification of the signal by enzyme cascades. This results when an enzyme associated with a signal receptor is activated. The activated enzyme in turn, catalyzes the activation of many molecules of a second enzyme, each of which activates many molecules of a third enzyme, and so on. Amplifications by several orders of magnitude can be produced in milliseconds by such cascades.

Transduction systems are MODULAR. Cells mix and match signaling proteins and molecules to create complexes with different functions and/or cellular locations. Signaling proteins have multiple binding domains allowing them to form complexes with numerous other proteins resulting in the formation of a wide variety of multienzyme complexes. A common theme is the binding of one protein to a phosphorylated domain on another; phosphorylation and dephosphorylation controls the interactions.

Transduction systems undergo DESENSITIZATION. A signal continuously present results in DESENSITIZATION (down regulation) of the receptor system. Phosphorylation and/or endocytosis renders the receptor unable to respond to the signal molecule. RESENSITIZATION occurs when the signal molecule concentration falls below a certain threshold level and the receptor is dephosphorylated and/or reinserted into the cell membrane.

Transduction systems are INTEGRATED. INTEGRATION is the ability of the system to receive multiple signals and produce a unified appropriate response Pathways converge at several levels resulting in multiple levels of cross talk.

Signal Molecules Hormones, Neurotransmitters & Eicosanoids

These are three major types of signal molecules; 1. Steroid Hormones 2. Modified Amino Acids, Peptides, or Proteins 3. Eicosanoids.

Neurotransmitters are often modified amino acids or small peptides. Hormones, growth factors, cytokines,


etc., (signal molecules) can be any of the three types. All of these information molecules require specific protein receptors to evoke a response within the cell. Receptor proteins specifically and avidly bind the signal molecule.

Steroid hormones are derivatives of cholesterol. They are nonpolar molecules that can cross the cell membrane without the aid of a transport protein. Once inside of the cell they bind to a specific receptor protein and the hormone/receptor complex migrates to the nucleus where it stimulates the expression of some gene or genes and inhibits the expression of other genes (Nuclear Receptor). The action of steroid hormones will be examined in more detail when the control of gene expression is discussed.

Modified amino acid, peptide, or protein signal molecules as well as the eicosanoids can not cross the cell membrane. They cannot cross the membrane because they are too large, too polar, or both. These signal molecules bind to specific receptor proteins. These receptors are integral membrane proteins containing a signal molecule binding domain on the exterior surface of the cell and a domain that interacts with the transduction system on the interior.

Information Transduction Systems

In general, six INFORMATION TRANSDUCTION SYSTEMS are employed by cells:

1. Steroid/Nuclear Receptors. These were briefly touched on above and will be discussed in more detail when the mechanisms controlling gene expression are discussed.

2. Gated ion channels. 3. Receptors associated with guanosine nucleotide binding proteins; trimeric G proteins or monomeric

G proteins. When the receptor is occupied by a signal molecule the trimeric or monomeric G protein is activated, that in turn activates an enzyme that produces a second messenger. The second messenger activates additional down stream enzymes to bring about the appropriate response.

4. Tyrosine Kinase receptors. These proteins contain two functional domains, an extracellular receptor domain for signal molecule binding and an intracellular tyrosine protein kinase domain. The extracellular receptor domain binds the signal molecule and binding activates the intracellular protein kinase domain. The active kinase transfers phosphate from ATP to specific tyrosine residues on substrate proteins.

5. Guanylyl Cyclase receptors. These proteins have two functional domains, an extracellular receptor domain for the signal molecule and an intracellular domain with guanylyl cyclase enzyme activity. Binding of the signal molecule activates the guanylyl cyclase domain that enzymatically converts GTP into the second messenger cyclic GMP (cGMP). The cGMP interacts with other proteins in the cell modulating their activity.

6. Adhesion receptors bind specific ligands and communicate with the cytoskeleton to bring about cell migration or cell adhesion to the extracellular matrix.

The final response to the signal molecule is either a change in cellular ion concentrations (ion channels, G proteins and/or tyrosine kinase receptors), a change in enzyme/protein concentration or composition (steroid receptors), or changes in cellular enzyme activity (G proteins, tyrosine kinase receptors, guanylyl cyclase receptors, adhesion receptors).


Gated Ion Channels The NICOTINIC ACETYLCHOLINE RECEPTOR is an example of a ligand gated ion channel. The receptor is a heteropentameric protein composed of 2α, 1β, 1γ, and 1δ subunit. Each of the 5 subunits have 4 transmembrane helical segments. In the resting state, a neuron has a -60 to -70 mV potential across the membrane (inside more negative) due to the high concentration of Na+ outside of the cell and the high concentration of K+ inside the cell. Each α subunit contains a binding site for acetylcholine. Acetylcholine binds to the α subunits in a cooperative manner, binding of the first makes binding of the second easier. When two acetylcholine molecules are bound to the receptor, the channel undergoes a “twisting” conformational change that moves five Leucine side chains (one from each subunit) out of the way, opening the channel so that Na+ or Ca2+ can enter the cell. The Na+ or Ca2+ move down the concentration gradient. If sufficient receptors are activated, the membrane becomes depolarized and voltage gated channels for K+ distal to the receptor are opened. The opening of the voltage gated channels propagates the impulse down the neuron or muscle cell.

Transduction Systems Associated with Heterotrimeric Guanosine Nucleotide Binding Proteins Heterotrimeric G Proteins

The Component Parts of the Gs and Gi System

All of the transduction systems coupled to HETEROTRIMERIC GUANOSINE NUCLEOTIDE BINDING PROTEINS have two components in common. The first component of these transduction systems is the Receptor Protein. All of the receptors associated with the Heterotrimeric Guanosine Nucleotide Binding Protein are intrinsic membrane proteins with seven transmembrane helical segments. These receptors are called SERPENTINE RECEPTORS because the protein “snakes” back and forth across the membrane seven times. They contain an extracellular domain that binds the “signal molecule” (hormone, neurotransmitter, growth factor, tastant, or odorant) and an intracellular domain that recognizes and interacts with the Heterotrimeric G Protein. When the signal molecule binds to the receptor, the receptor undergoes a conformational change that is communicated to the associated guanosine nucleotide binding protein.

The second component common to all of these signal transduction systems is a group of related proteins called the HETEROTRIMERIC GUANOSINE NUCLEOTIDE BINDING PROTEINS. The Heterotrimeric Guanosine Nucleotide Binding Proteins are a subset of the superfamily of Guanosine Nucleotide Binding Proteins, the G Proteins. As the name implies, the heterotrimeric G proteins is composed of three subunits; an α subunit, a β subunit, and a γ subunit. There are 20 different α subunits, 6 different β subunits and 12 different γ subunits. By mixing and matching these subunits a large number of different heterotrimers can be formed


each with a slightly different specificity or activity. There are approximately 800 different receptors coupled with heterotrimeric G proteins. The α and γ subunits are lipid linked {lipid anchored} membrane proteins bound to the inner leaflet of the cell membrane; the β subunit is tightly bound to the γ.

The α subunit of the heterotrimeric guanosine nucleotide binding protein binds guanosine-5´-diphosphate {GDP} or guanosine-5´-triphosphate {GTP} with nearly equal affinity. When GDP is bound, the α subunit is in the “inactive” or “unstimulated” state. The inactive α subunit has high affinity for its corresponding β and γ subunits, it binds to them tightly, and this trimeric G protein complex binds tightly to the intracellular domain of the receptor. When GTP is bound to the α subunit it is in the “active” or “stimulated” state. Binding of GTP activates the α subunit and the α subunit-GTP complex dissociates from the βγ complex and from the receptor. Without the α subunit bound, the βγ complex also dissociates from the receptor. The “active” α subunit-GTP complex binds to / interacts with membrane bound enzymes/proteins modulating their activity. The α subunit has a slow GTPase (GTP hydrolase) activity. It slowly hydrolyzes bound GTP to GDP and PO4–3. When the α subunit hydrolyzes GTP to GDP it converts itself from the “active” to the “inactive” state. The α subunit is a SELF LIMITING TRANSDUCTION ELEMENT meaning it turns itself “off” by hydrolyzing the bound GTP to GDP. Upon hydrolysis of the GTP to GDP the α subunit-GDP complex dissociates from the enzymes/proteins it has affected and migrates back to its receptor where it (re)binds the βγ subunits and (re)binds to its receptor.

Since the affinity of the α-subunit for GTP and GDP are nearly equal there are proteins within the cell that interact with the G proteins to modulate their activity. GUANOSINE NUCLEOTIDE-EXCHANGE FACTORS interact with the trimeric G proteins and speed up the intrinsically slow process of replacing bound GDP with GTP. GTPASE ACTIVATOR PROTEINS (GAPs) bind to and interact with the α subunit and stimulate the GTPase activity of this subunit. The GAPs contribute an Arg side chain necessary for GTP hydrolysis and therefore modulate how long the switch will remain “on”.

The proteins and enzymes described below are components of the Gs, and Gi coupled transduction systems. The structure and function of these proteins will be described and then the transduction system will be assembled and examined.

An important protein of the Gs and Gi transduction systems is the enzyme Adenylate Cyclase (Adenylyl Cyclase). This enzyme is an intrinsic membrane protein. It catalyzes the conversion of ATP to 3´,5´-Cyclic AMP (cAMP) and pyrophosphate (P2O7–4). cAMP belongs to a group of molecules called SECOND MESSENGERS. This enzyme displays an intrinsic baseline activity within the cell. Its activity is modulated by the Gsα-GTP complex and/or the Giα-GTP complex.

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The enzyme Cyclic Nucleotide Phosphodiesterase (Phosphodiesterase) catalyzes the hydrolysis of cAMP to adenosine-5’-monophosphate, rendering cAMP inactive as a second messenger. This enzyme is always active within the cell.

The enzyme cAMP Dependent Protein Kinase (Protein Kinase A or PKA) is an allosteric enzyme. The inactive form is a tetramer composed of two Regulatory Subunits and two Catalytic Subunits. It is converted to the active form when 2 cAMP molecules bind to each of the two regulatory subunits; a total of four cAMP must bind to activate the enzyme. cAMP binding causes a conformational change in the regulatory subunits resulting in the release and activation of the catalytic subunits. The active catalytic subunits catalyze the transfer of phosphate (–PO3–2) from ATP to a specific set of cellular proteins or enzymes. A small specific set of cellular proteins are substrates for cAMP Dependent Protein Kinase. Phosphate is covalently linked to the protein by an ester bond to the hydroxyl group of particular serine or threonine side chains. Protein kinase A is a serine/threonine protein kinase.

Phosphoprotein Phosphohydrolases (Phosphoprotein Phosphatases or the Protein Phosphatases {synonyms}) are a group of enzymes that catalyze the hydrolytic removal of phosphate from phosphorylated proteins. With the phosphate removed these proteins return to their original state of activity.

ADAPTOR PROTEINS play a role in localizing the signaling response to a specific region of the cell. ADAPTOR PROTEINS belong to the scaffold functional class of proteins. They have binding sites for two or more components of the signaling system (they are multivalent). When adaptor proteins bind to the components of a signaling system a closely associated functional unit is formed. Adaptor proteins have been identified as part of many/most of the transduction systems. Adaptor proteins that interact with Protein Kinase A of the Gs and Gi transduction systems are some of the best characterized. These proteins, called A KINASE ANCHORING PROTEINS (AKAP), localize Protein Kinase A to regions of the cell where its activity is important, such as microtubules, actin, the mitochondria, the nucleus, etc. AKAP79 binds Protein Kinase A (PKA) to Adenylate Cyclase, this interaction effective activates PKA; whereas AKAP250 binds PKA to Cyclic Nucleotide Phosphodiesterase, the interaction would rapidly inactivate PKA.

By combining the different G proteins with different receptors and the enzymes described above, transduction systems that produce a variety of responses to signal molecules can be obtained.

The Gs Effector System

The Gs effector system consists of the serpentine receptor protein, the trimeric Gs protein complex, Adenylate Cyclase, Protein Kinase A (cAMP dependent protein kinase or PKA), Phosphodiesterase, and Protein Phosphatases. In the absence of signal molecule, GDP is bound to the α subunit of the Gs protein, the β and γ subunits are associated with the α subunit, and the Gs protein complex is bound to the intracellular domain of the serpentine receptor (Gs System Panel 1). Adenylate Cyclase is functioning at its baseline level, converting ATP to cAMP at a slow constant rate. The cAMP produced enters a small cytoplasmic pool of cAMP present in the cell. Much of this pool of cAMP is rapidly destroyed by cAMP Phosphodiesterase. The fraction that escapes destruction stimulates a low level of Protein Kinase A activity. The enzymes/proteins phosphorylated by PKA function at their alternative level of activity (stimulated or inhibited by reversible covalent modification) until Phosphoprotein Phosphohydrolase removes the


phosphate group whereupon they return to their baseline activity. This constant interplay of proteins and enzymes maintains the cell in its baseline or resting metabolic state.

When the signal molecule binds to its receptor, it induces a conformational change in the receptor that is transmitted to the Gs protein. The Gs protein undergoes a conformational change which, with the aid of the GUANOSINE NUCLEOTIDE-EXCHANGE FACTORS, GDP dissociates from the α subunit, GTP to bind the α subunit, and the Gsα-GTP complex to dissociate from the β and γ subunits and from the receptor (Gs System Panel 2).

The Gsα-GTP complex migrates along the inner surface of the cell membrane until it encounters Adenylate Cyclase. The Gsα-GTP complex binds to Adenylate Cyclase and simulates its activity. Stimulated Adenylate Cyclase catalyzes theformation of cAMP at a greatly increased rate. Within seconds the intracellular concentration of cAMP increases 1000 to 10000 fold above baseline levels even with Phosphodiesterase hydrolyzing the cAMP to AMP at nearly Vmax (Gs System Panel 3). The massive increase in cAMP concentration allows for an increased amount of cAMP to bind to the regulatory subunits of Cyclic AMP Dependent Protein Kinase (PKA). cAMP binding activates PKA by causing the dissociation of the regulatory subunits from the catalytic subunits. The active protein kinase A phosphorylates its specific set of substrate proteins and enzymes. The phosphorylated molecules are either stimulated by this reversible covalent modification or they are inhibited depending upon the particular enzyme phosphorylated. This change in enzyme activity (a specific set of enzymes stimulated by reversible covalent modification and/or inhibited) is the ultimate cellular response to the outside stimulus, to the signal molecule.

The hormone response is turned off by four mechanisms (Gs System Panel 4):

1. The signal molecule must be removed from the receptor. As long as signal molecule is present on the


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receptor, the system will be in its stimulated state. Gsα subunit will hydrolyze GTP to GDP, but as soon as the Gsα-GDP complex interacts with the occupied receptor the GDP is exchanged for GTP and the Gsα subunit is reactivated. Signal molecule removal is accomplished by one of three mechanisms. Either the signal molecule is destroyed by enzymes in the extracellular space, the signal molecule is taken up by the cell that secreted it (re-uptake), or the signal molecule-receptor complex is internalized by the cell. Once in the cell the signal molecule is destroyed and the receptor reinserted into the membrane.

2. The slow hydrolysis of GTP to GDP by the αsubunit of the G protein ± GTPASE ACTIVATOR PROTEINS, renders the Gsα subunit inactive. With GDP bound it no longer stimulates Adenylate Cyclase, the Gsα subunit-GDP complex dissociates from Adenylate Cyclase and (re)binds to the β and γ subunits and to its empty receptor.

3. Hydrolysis of cAMP to AMP by Phosphodiesterase shifts the equilibrium between the free form of cAMP and the form bound to PKA toward the free from. As cAMP dissociates from the regulatory subunit, the regulatory subunit rebinds the catalytic subunit returning Protein Kinase A to its baseline level of activity.

4. Hydrolysis of the PO4–3 from the phosphorylated enzymes by Protein Phosphatases brings the activity of these enzymes back to their “original baseline activity”. The Protein Phosphatases are always active within the cell.

NOTE: Signal molecules acting through the Gs effector system include: Corticotropin (ACTH), Corticotropin-Releasing Hormone (CRH), Dopamine, Epinephrine (β-receptors) {increases blood glucose concentration}, Follicle Stimulating Hormone (FSH), Glucagon {increases blood glucose concentration}, Histamine (H-2 receptor), Luteinizing Hormone (LH), Melanocyte Stimulating Hormone (MSH), Odorants (sense of smell), Parathyroid Hormone, Prostaglandins, Serotonin (5-HT-1a & 5-HT-2 receptors), Somatostatin, Tastants (bitter & sweet), and Thyroid Stimulating Hormone.


cyclic-AMP(cAMP)

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The Gi Effector System

The effector system that is coupled to the Gi protein utilizes the same component parts as the Gs system, but the effects are opposite to those observed with the Gs effector system. The Gi system inhibits the activity of Adenylate Cyclase resulting in a decrease in cytoplasmic cAMP concentrations.

When the signal molecule binds to the receptor, the receptor undergoes a conformational change that is communicated to the Gi protein. The Gi protein undergoes a conformational change, GDP is exchanged for GTP on the Giα subunit, and the Giα-GTP complex dissociates from the β and γ subunits and from the receptor. The Giα-GTP complex migrates along the inner surface of the membrane until it encounters Adenylate Cyclase, binds to it, and inhibits its activity. With the activity of Adenylate Cyclase inhibited, the cellular concentration of cAMP decreases below baseline levels. The decrease is directly due to the action of Phosphodiesterase which is always actively hydrolyzing cAMP to AMP. As the cellular concentration of cAMP decreases, the activity of cAMP Dependent Protein Kinase (PKA) decreases below its baseline level since it requires cAMP for activity. The decreased activity of PKA coupled with the constant activity of Phosphoprotein Phosphatase results in a decreased concentration of phosphorylated cellular enzymes/proteins. The decrease in phosphorylated cellular proteins with its concomitant change in cellular enzyme activity is the ultimate cellular response. The removal of PO4–3 activates some enzymes/proteins and inactivates others.

The hormone response is turned off by the same four mechanisms that turn off the Gs system.

NOTE: The Gi effector system is coupled to the α2 receptor of norepinephrine. It also mediates the action of the opiates, some of the prostaglandins, somatostatin, and angiotensin.

Hepatocytes contain β-receptors for epinephrine coupled to the Gs effector system as well as α2 receptors for epinephrine coupled to the Gi system. The cell is able to respond to both, how is this possible. One possible explanation is the relative affinities of the receptors for epinephrine. One receptor, probably the β has a significantly higher affinity then the α2. With this arrangement the cell would respond via the β-receptors at low hormone concentrations and respond via the α2 receptors at high hormone concentrations. A second possible explanation is cross talk between the systems. The Giβγ complex has been shown to bind to the Gsα-GTP complex preventing it from binding to and stimulating Adenylate Cyclase. Both of these possibilities are supported by the observation that liver cell membranes contain far more Gi than Gs. The activation of Gi in these cells would therefore release sufficient Giα-GTP complex to bind to and inhibit


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Adenylate Cyclase as well as enough Giβγ to bind to and inhibit the available Gsα-GTP complex.

The Component Parts of the Gq System

The first component of this transduction systems is the Serpentine Receptor Protein. All of the receptors associated with the Heterotrimeric Guanosine Nucleotide Binding Protein are intrinsic membrane proteins with seven transmembrane helical segments.

The second component of this signal transduction systems is the HETEROTRIMERIC GUANOSINE NUCLEOTIDE BINDING PROTEINS. The Heterotrimeric Guanosine Nucleotide Binding Protein in this case is composed of the Gq α subunit, and β, and γ subunits different from the Gs and Gi.

Since the affinity of the α-subunit for GTP and GDP are nearly equal there are proteins within the cell that interact with the G proteins to modulate their activity. GUANOSINE NUCLEOTIDE-EXCHANGE FACTORS interact with the trimeric G proteins and speed up the intrinsically slow process of replacing bound GDP with GTP. GTPASE ACTIVATOR PROTEINS (GAPs) bind to and interact with the α subunit and stimulate the GTPase activity of this subunit. The GAPs contribute an Arg side chain necessary for GTP hydrolysis and therefore modulate how long the switch will remain “on”.

A membrane bound ISOENZYME of Phospholipase C plays a role in several transduction systems. ISOENZYMES have different primary structures but they catalyze identical chemical reactions. Since they catalyze identical reactions there is a large amount of structural homology among isoenzymes. Isoenzymes are often tissue specific or specific to a developmental epoch. Although they catalyze the same reaction, they may be specific for different substrates, have very different Km’s for several different closely related substrates, or they may catalyze the reaction at very different rates. Phospholipase C in general catalyzes the hydrolysis of the bond between glycerol and phosphate in the phosphoglycerides. There are several different isoenzymes of Phospholipase C. Soluble forms of this enzyme are used in the digestive process and in membrane remodeling. The isoenzymes of interest here are Phospholipase C-β and Phospholipase C-γ. These isoenzymes are intrinsic membrane proteins with absolute substrate specificities. Phospholipase C-β and Phospholipase C-γ will only utilize the membrane phosphoglyceride Phosphatidylinositol-4,5-bisphosphate (PIP2) present in the inner leaflet of the membrane as a substrate. The products of the hydrolysis reaction are Diacylglycerol (DAG, glycerol with fatty acids on C-1 and C-2) and Inositol-1,4,5-trisphosphate (IP3). DAG and IP3 are SECOND MESSENGERS.

Protein Kinase C (Calcium Activated Protein Kinase or PKC) exists as an inactive soluble cytoplasmic form and an active membrane bound form. PKC is an AMPHITROPIC PROTEIN. The inactive form is activated when it


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binds four Ca+2 and the protein-Ca+2 complex is bound by diacylglycerol (DAG) in the cytoplasmic membrane. Calcium ions serve as a bridge between the protein and the negatively charged heads of neighboring membrane phosphoglycerides such as phosphatidylserine. This anchors the enzyme to the membrane as an extrinsic membrane protein. Once bound to the membrane DAG, present in the membrane, binds to the enzyme and completes the activation process. In the absence of one or both of these components the enzyme is cytoplasmic and inactive. As a protein kinase, this enzyme catalyzes the transfer of phosphate (–PO3–2) from ATP to a specific set of cellular proteins and/or enzymes; only a small unique set of proteins are substrates for Calcium Activated Protein Kinase. Phosphate is covalently linked to the protein by an ester bond between the phosphate and the hydroxyl group of a serine or threonine side chain. Protein kinase C is a serine/threonine protein kinase.

The Ca+2-Calmodulin Protein Kinases are another of a group of protein kinases that can be activated by transduction systems. CALMODULIN (CALcium ion MODULating proteIN) is a cytoplasmic protein that binds calcium ions with high affinity. The Ca+2-Calmodulin complex then binds to other cellular proteins changing their activity. One protein that binds the Ca+2-Calmodulin complex is the Ca+2-Calmodulin Protein Kinases. When the Ca+2-Calmodulin complex binds to these protein kinases their activity is stimulated, they are “turned on”. The Ca+2-Calmodulin protein kinases are serine/threonine protein kinases.

Phosphoprotein Phosphohydrolases (Phosphoprotein Phosphatases or the Protein Phosphatases {synonyms}) are a group of enzymes that catalyze the hydrolytic removal of phosphate from phosphorylated


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proteins. With the phosphate removed these proteins return to their original state of activity.

The Gq Effector System

The Gq effector system does not utilize adenylate cyclase and protein kinase A, rather it utilizes Phospholipase C-β, Protein Kinase C and possibly other protein kinases (Ca+2-Calmodulin Protein Kinases) and proteins/enzymes to mediate the response. The smooth endoplasmic reticulum also plays a role in this transduction system. In the unstimulated state the receptor is empty, the Gq protein is associated with the receptor because GDP is bound, Phospholipase C-β and Protein Kinase C are inactive, and the smooth endoplasmic reticulum is actively sequestering Ca2+ (Gq System Panel 1 [above]).

The signal molecule binds to the receptor and causes a conformational change that is communicated to the Gq protein which also undergoes a conformational change (Gq System Panel 2 [above]). The conformational change in the Gq initiates the GDP for GTP exchange and the Gqα-GTP complex dissociates from the β and γ subunits and from the receptor. The Gqα-GTP complex migrates along the inner leaflet of the membrane until it encounters Phospholipase C-β. Phospholipase C-β is activated when the Gqα-GTP complex binds to it (Gq System Panel 3). Phosphatidylinositol-4,5-bisphosphate (PIP2), the substrate of Phospholipase C-β, is a membrane phosphoglyceride present in the inner leaflet. It is brought to the activated enzyme by the constant lateral movement of the membrane lipids. The Phospholipase C-β cleaves phosphatidylinositol-4,5-bisphosphate into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). The IP3 migrates in the cytosol (it is too polar to remain in the membrane) to the smooth endoplasmic reticulum (Gq System Panel 4). The IP3


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binds to the signal gated Ca+2 channel in the smooth endoplasmic reticulum, opens the gate of the channel, and releases Ca+2 into the cytosol, IP3 is the second messenger that opens the gate. The increased cytosolic Ca+2 concentrations produces a variety of effects.

For some cell types the release of Ca+2 from the sER is sufficient for a response. In skeletal muscle the first step in the contraction process is the release of Ca+2 from the sarcoplasmic reticulum (a specialized form of smooth ER). The release of Ca+2 is also important for cells that secrete other signal molecules. Ca+2 is necessary for the rearrangement and re-polymerization of cyctoskeletal elements that direct the movement of secretory granules from the Golgi to the cytoplasmic membrane for release. These Ca+2 mediated processes fall under the heading of SECRETION-CONTRACTION COUPLING.

Calcium ions bind to Protein Kinase C and anchors it to the membrane (Gq System Panel 5). Diacylglycerol, the other product, the other second messenger of Phospholipase C-β, migrates along the inner leaflet the cell membrane until it encounters Protein Kinase C. DAG binds tightly to Protein Kinase C. When Ca+2 and diacylglycerol are bound to Protein Kinase C this enzyme is activated and it phosphorylates a specific set of cellular enzymes/proteins. These phosphorylated enzymes/proteins have increased or decreased enzymatic activity (depending upon the specific enzyme phosphorylated) and it is the change in enzymatic activity that is the ultimate cellular response to the hormone / outside stimulus. Some of the proteins phosphorylated by PKC is the Ca+2 active transporter of the plasma membrane and the Ca+2 channel protein of the ER. Phosphorylation of the channel proteins prolong the response of the Gq system by controlling the transport of Ca+2 into and out of the cell.


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The elevated Ca+2 levels in the cell can also activate the Ca+2-Calmodulin Protein Kinases, if they are present in the cell (Gq System Panel 6). The Ca+2-Calmodulin Protein Kinases have a different target set of enzymes/proteins as substrates. When these proteins are phosphorylated an additional or a different set of cellular responses is observed. Elevated Ca+2 levels in the cell can also activate Nonspecific (Calcium Activated)Protein Kinases that have a third different set of enzymes/proteins as substrates

One of the enzymes that may be activated by Protein Kinase mediated phosphorylation is an isoenzyme of Phospholipase A2. Quite often the fatty acid attached to carbon two of the diacylglycerol produced by Phospholipase C-β is arachidonate. The arachidonate is released from the diacylglycerol by the hydrolytic action of the activated Phospholipase A2 and once released it is used as a precursor for the synthesis of one or more of the eicosanoids. It appears that utilization of the Gq effector system in some cell types stimulates the synthesis of one or more of the eicosanoids. The ultimate, final cellular response observed when the Gq effector system is activated can be due to the activity of Protein Kinase C, the Ca+2-Calmodulin Protein Kinases, the Nonspecific Protein Kinases, the eicosanoids (if Phospholipase A2 is activated), or any combination of these (Gq System Panel 7).

There is cross-talk between the Gq transduction system and the Gs / Gi systems. In some tissues both Adenylyl Cyclase and Phosphodiesterase are stimulated by Ca+2. The increased concentration of Ca+2 brought about by the Gq system can bring about rapid transient changes in cAMP concentrations thereby effecting the activity of PKA.


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The activity of the Gq effector system is turned off by a variety of mechanisms (Gq System Panel 8):

1. The signal molecule is removed from the receptor as described above. 2. The slow GTPase activity of the Gqα subunit, ± GTPASE ACTIVATOR PROTEINS, converts the bound GTP

to GDP and PO4–3 rendering the Gqα subunit inactive. 3. The Ca+2 channel is closed when inositol-1,4,5-trisphosphate is converted to inositol by the action of

inositolphosphate phosphatases present in the cytoplasm. 4. Ca+2 is “pumped” the back into the endoplasmic reticulum by primary active transport proteins, Ca+2

ATPases. Accumulation of Ca+2 by the ER rapidly lowers cytosolic Ca+2 concentrations. 5. The decrease in intracellular Ca+2 turns Protein Kinase C and the other Ca+2 controlled protein kinases

off. 6. Diacylglycerol is converted back to a phosphoglyceride which also turns off Protein Kinase C.

Additional enzymes catalyze the synthesis of Phosphatidylinositol-4,5-bisphosphate from Phosphatidylinositol.

7. The PO4–3 is removed from the phosphorylated proteins by the action of the Protein Phosphatases returning them to their baseline activity.

NOTE: Hormones / Neurotransmitters acting through the Gq effector system include: Acetylcholine (muscarinic M1 receptor), Epinephrine (α1-receptors), Angiogenin, Angiotensin II, ATP, Auxin, Gastrin Releasing Peptide, Glutamate, Gonadotropin Releasing Hormone (GRH), Histamine (H-1 receptor), Oxytocin, Platelet Derived Growth Factor, Serotonin (5-HT-1c receptor), Thyrotropin Releasing Hormone (TRH), and Vasopressin.

Tyrosine Kinase Receptor Systems

The Tyrosine Kinase part of these receptors catalyzes the transfer of phosphate from ATP to specific tyrosine residues on the proteins and enzymes that are substrates for these receptor kinases. Protein kinase A, protein kinase C, and the Ca+2-calmodulin protein kinases are serine/threonine protein kinases. They transfer phosphate from ATP to specific serine or threonine residues on their substrate proteins. As with the other kinases the proteins and enzymes phosphorylated by the tyrosine kinases exhibit a change in activity.

Many of the tyrosine kinase receptor systems also contain one or more of the monomeric G proteins as part of the enzymatic cascade. Monomeric G proteins are single polypeptide chains that can be bound by either GDP (inactive form) or GTP (active form). The α subunit of the trimeric G proteins is structurally and functionally very similar to the monomeric G proteins, suggesting an evolutionary relationship. The insulin receptor system will be examined in some detail as an example of a system that contains both a tyrosine kinase and a monomeric G protein.

The Insulin Receptor

The receptor-effector system for the hormone INSULIN is one example of a tyrosine kinase receptor system. Insulin has many diverse effects upon cellular metabolism. Insulin is released from the β cells of the pancreas in response to elevated blood glucose levels. The effects of insulin include:

1. increased uptake of glucose from the blood by the insulin dependent tissues using the GluT4 passive


glucose transporter. 2. increased utilization of glucose for cellular energy. 3. increased storage of glucose as glycogen. 4. increased synthesis of fatty acids and storage of triacylglycerols. 5. increased protein synthesis. 6. increased cell division.

All of these effects are mediated by the insulin tyrosine kinase effector system.

The insulin receptor is a tetramer composed of 2 α subunits, the insulin binding sites, and 2 β subunits, the tyrosine kinase activity. Insulin binds to the α subunits in a cooperative manner and the binding of 2 insulin molecules to the receptor causes a conformational change in the 2 β subunits partially activating the Tyrosine Kinase. One partially activated β subunit phosphorylates its partner at three sites (tyr side chains) and this cross phosphorylation of the β dimer opens the substrate/active site of the Tyrosine Kinases so it can bind the other substrate proteins. The cross phosphorylation also prolongs the response to insulin. Insulin can be removed from the fully active receptor and the response will be sustained as long as the receptor remains phosphorylated. The fully active Tyrosine Kinase phosphorylates a variety of cellular proteins to bring about the diversity of cellular responses.

The cascade leading to increased protein synthesis and/or increased cell division is as follows:

1. The active insulin receptor Tyrosine Kinase phosphorylates Insulin Receptor Substrate-1 (IRS-1). 2. The phosphotyrosine of IRS-1 is bound by the SH2 (Src Homology 2) domain of Grb2. Several

signaling proteins contain the SH2 domain and the SH2 domain of these proteins all bind phosphotyrosine residues on specific activating proteins.

3. The Grb2•PO4-IRS-1 complex then binds Sos to form a Sos•Grb2•PO4-IRS-1 complex. Grb2•PO4-IRS-1 is a ADAPTOR PROTEIN for Sos.

4. The Sos•Grb2•PO4-IRS-1 complex binds to Ras (Rat Sarcoma virus protein) and Sos acts as a GUANOSINE NUCLEOTIDE-EXCHANGE FACTOR to stimulate the dissociation of GDP from Ras and the binding of GTP. Ras is a monomeric G protein. It is similar in structure and function to the α subunit of the heterotrimeric G proteins. Ras can bind GDP or GTP and it slowly catalyzes the hydrolysis of GTP to GDP and phosphate. Ras is a member of the G Protein Superfamily.

5. With GTP bound, Ras can activate the protein kinase Raf-1, the first of three protein kinases in this

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cascade. (Raf-1 is also called Mitogen Activated Protein Kinase Kinase Kinase.) 6. The protein kinase MEK (Mitogen-activated ERK-activating Kinase; Mitogen Activated Protein Kinase

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8. Phosphorylated, active MAPK migrates to the nucleus where it phosphorylates proteins such as Elk-1. 9. The phosphorylated Elk-1 binds to SRF (Serum Response Factor) and this complex modulates the

transcription and expression of about 1000 insulin sensitive genes.


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The cascade leading to increased glucose uptake and increased glycogen synthesis is as follows:

1. The active insulin receptor Tyrosine Kinase adds phosphate to Insulin Receptor Substrate-1 (IRS-1). 2. The phosphotyrosine of IRS-1 is bound by the SH2 domain of Phosphotidylinositol-3-Kinase (PI-3K). 3. When PI-3K is bound to PO4-IRS-1 it is an active LIPID KINASE. It catalyzes the transfer of a phosphate

from ATP to phosphatidylinositol-4,5-bisphosphate (PIP2) to form ADP and phosphatidylinositol-3,4,5 trisphosphate (PIP3).

4. Phosphatidylinositol-3,4,5-trisphosphate molecules are binding sites for Protein Kinase B (PKB) on the membrane. {Protein Kinase B is an AMPHITROPIC PROTEIN} When bound to the membrane Protein Kinase B is phosphorylated by the Protein Kinase PDK-1 converting it from an inactive enzyme to an active protein kinase.

5. The active Protein Kinase B catalyzes the transfer of phosphate from ATP to Glycogen Synthase Kinase 3 (GSK3) converting this from an active protein kinase to an inactive form. The normal substrate for Glycogen Synthase Kinase 3 is the enzyme Glycogen Synthase. Glycogen Synthase is the enzyme that catalyzes the formation of the α1 → 4 glycosidic bonds in glycogen, it catalyzes the synthesis of the majority of the glycogen molecule.

6. In the absence of insulin the phosphorylation of Glycogen Synthase by Glycogen Synthase Kinase 3, converts Glycogen Synthase to a less active enzyme, and glycogen synthesis is very much inhibited.

7. In the presence of insulin Glycogen Synthase Kinase 3 is rendered inactive by the action of Protein Kinase B and Glycogen Synthase Kinase 3 is no longer able to phosphorylate Glycogen Synthase.

8. The always active Phosphoprotein Phosphatases within the cell remove the phosphate from Glycogen Synthase, shifting the enzyme from the inactive to the active form of Glycogen Synthase, and the rate of glycogen synthesis increases dramatically.

9. The active Protein Kinase B also stimulates the movement of glucose transporters (GluT4) from their sequestered sites on membrane vesicles within the cytoplasm to the plasma membrane. The increased number of glucose transporters in the cell membrane stimulates the cellular uptake of glucose from the blood.

Other signal molecules, especially growth factors, that function via Tyrosine Kinase Receptors and the MAPK cascade include Vascular Epidermal Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF), and Fibroblast Growth Factor (FGF).

The Δ9-tetrahydrocannabinol (THC) and the endocannabinoids, e.g., anandamide, function via the CB1 receptor in the brain to activate a MAPK cascade as well as Protein Kinase B.

There is cross talk between the insulin receptor tyrosine kinase and the β-receptors for Epinephrine. One of the effects of Insulin is to stimulate the uptake of glucose from the blood post meal and the storage of glucose as glycogen in the insulin dependent tissues (skeletal muscle is insulin dependent). The β-receptors of Epinephrine stimulate the release of glucose from glycogen in muscle to power contraction during the fight or flight response. If both hormones are present in the circulation a futile cycle within the skeletal muscle would result - insulin storing glucose and epinephrine releasing it. Insulin and its receptor tyrosine kinase prevents the futile cycle by directly interacting with the β-receptors for Epinephrine. The tyrosine kinase when fully active phosphorylates 2 tyrosine residues near the C terminal end of the β-receptor and Protein Kinase B phosphorylates 2 serine residues near the C terminal end of the receptor. Phosphorylation


of the β-receptors for Epinephrine at these four sites stimulates the internalization of the receptors and limits / prevents the skeletal muscles response to Epinephrine.

Receptor Guanylyl Cyclases, cGMP, and Protein Kinase G

Receptor enzymes that when activated convert GTP to guanosine-3´,5´-cyclic monophosphate (cGMP) fall into the Guanylyl Cyclase class of receptors. These receptors are single protein molecules with three domains; an extracellular domain that binds the signal molecule, an single trans membrane α-helical domain, and an intracellular Guanylyl Cyclase domain. The signal molecule when bound to the extracellular domain activates the Guanylyl Cyclase domain. Guanylyl Cyclase catalyzes the conversion of GTP to cGMP. cGMP, a second messenger, that when present allosterically activates cGMP-Dependent Protein Kinase (Protein Kinase G or PKG). The regulatory and catalytic domains of PKG reside on a single protein molecule, unlike PKA. In the kidney these receptors cause increased secretion of Na+ and H2O; in the intestine they control the secretion of Cl–; in heart muscle they signal relaxation; and in the brain they may play a role in development and adult brain function.

Cancer and the Signal Transduction Systems Oncogenes

Tumors and cancer are the result of uncontrolled cell division. Normally cell division is controlled by a family of extracellular proteins called GROWTH FACTORS. A different set of signal molecules secreted by Leukocytes, the CYTOKINES, stimulate cell division under the conditions of inflammation and/or tissue damage. The actions of GROWTH FACTORS and CYTOKINES are mediated by Signal Transduction Systems. Some of the known carcinogens mimic the structure and actions of a second messenger. For example the PHORBOL ESTERS mimic the action of Diacylglycerol on Protein Kinase C, activating this enzyme under incorrect conditions resulting

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in abnormal cell growth. ONCOGENES were originally discovered in tumor causing viruses, then later found to be very similar to or derived from genes present in the host cells. Apparently during viral infection a segment of the host genome was incorporated into the viral genome. At some point the host gene that was incorporated into the viral genome became defective and when this defective gene is expressed in the host cell the abnormal viral protein product interferes with normal cell growth and differentiation, sometimes resulting in a tumor. There are oncogenes that encode for defective signal molecules, growth factors, cytokines, receptors, G proteins, protein kinases, and nuclear transcription factors. Some oncogenes encode tyrosine kinase receptors with defective or missing signal binding sites so that the tyrosine kinase activity is unregulated, always “on”. In addition mutant forms of the G protein Ras are common in tumor cells. The Ras oncogene encodes a protein with normal GTP binding but no GTPase activity. This mutant Ras protein is therefore always “on” regardless of the signals coming from normal receptors. The result of either mutation / oncogene can be unregulated cell growth.


Biosignaling - Marquette University · Gated Ion Channels The NICOTINIC ACETYLCHOLINE RECEPTOR is...

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Transcript of Biosignaling - Marquette University · Gated Ion Channels The NICOTINIC ACETYLCHOLINE RECEPTOR is...