Biochem SG 12
Why is ligand binding being reversible, ligand having both a Ka and Kd.
- The affinity between signal (ligand) and receptor can be expressed as the dissociation constant Kd, usually 1010 M or less—meaning that the receptor detects picomolar concentrations of a signal molecule. Receptor-ligand interactions are quantified by Scatchard analysis, which yields a quantitative measure of affinity (Kd) and the number of ligand-binding sites in a receptor sample. Receptor-ligand binding can be described by the following equilibrium equation R + L <-> RL Ka is the association constant and is calculated by looking at [RL]/[R][L]. -Ka describes the equilibrium between the complex and the unbound components of the complex. The association constant provides a measure of the affinity of the protein for the ligand. -Kd is the equilibrium constant for the release of the ligand. It is equal to the molar concentration of ligand at which half of the available ligand-binding sites are occupied. The more tightly a protein binds a ligand, the lower the concentration of ligand required for half the binding sites to be occupied, and thus lower the value of dissociation. -Ligand binding is reversible because in certain parts of the cell the ligand has to be released, while in other areas the ligand has to be interact and be delivered by the specific receptor so in order for both processes to occur, the interaction must be reversible.
Describe the general structure and domains of Adenylate Cyclase.
-AC is an integral protein of the plasma membrane, with its active site on the cytosolic face. It catalyzes the synthesis of cAMP from ATP. It consists of two bundles of six transmembrane segments. -Two catalytic domains extend as loops into the cytoplasm. A soluble (non-membrane bound) form of adenylyl cyclase has recently been characterized in mammalian sperm. This form of the enzyme appears to be activated by bicarbonate ion . -The associate of active Ga with adenylyl cyclase stimulates the cyclase to catalyze cAMP synthesis from ATP, raising the cytosolic [cAMP]. The interaction between Ga and adenylyl cyclase is possible only when Ga is bound to GTP.
Explain the function of the scaffold proteins AKAP
-AKAPs (A kinase anchoring proteins) are multivalent adaptor proteins where one part binds to the R subunits of PKA and another to a specific structure in the cell. this confines the PKA to the vicinity of that specific cell structure. -AKAPs act as a signaling hub that assembles signaling proteins on a scaffold that itself targets to various domains, such as the nucleus, in cells. This allows specific targeting of substrates to be regulated by phosphorylation (by PKA) and dephosphorylation (by phosphatases). The dimerization and docking (D/D) domain of the regulatory subunit dimer of PKA binds with the A-kinase binding (AKB) domain (an amphipathic helix) of AKAP. -(ex: specific AKAPs bind PKA to microtubules, actin filaments, Ca2+ channels, mitochondria, and the nucleus)
Explain signal transduction amplification of signal.
-Amplification by enzyme cascades results when an enzyme associated with a signal receptor is activated and, 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. Such cascades can produce amplifications of several orders of magnitude within milliseconds. -The response to the single must be also terminated such that the downstream effects are in proportion to the strength of the original stimulus. When enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade.
How does ubiquination of cyclin effect the activity of CDKs?
-CDK activity in regulated throughout the cell cycle by highly specific and precisely timed proteolytic breakdown of mitotic cyclins. The protein DBRP (destruction box recognizing protein) recognizes the sequence on the cyclin (that targets them for destruction) and initiates the process of cyclin degradation by bringing together the cyclin and another protein, called ubiquitin. Ubiquitin ligase covalently joins the ubiquitin and the cyclin, and several more ubiquitin molecules are then added on, which provides the signal for a proteolytic enzyme complex, or proteasome, to degrade cyclin. -Increased CDK activity activates cyclin proteolysis. Newly synthesized cyclin associates with and activates CDK, which phosphorylates and activates DBRP. Active DBRP then causes proteolysis of cyclin. Lowered [cyclin] causes a decline in CDK activity, and the activity of DBRP also drops through slow, constant dephosphorylation and inactivation by a DBRP phosphatase. The cyclin level is ultimately restored by synthesis of new cyclin molecules.
How is the JAK-STAT pathway unique?
-Cellular responses to dozens of cytokines and growth factors are mediated by the evolutionarily conserved Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway. These responses include proliferation, differentiation, migration, apoptosis, and cell survival, depending on the signal, tissue, and cellular context. JAK/STAT signaling is essential for numerous developmental and homeostatic processes, including hematopoiesis, immune cell development, stem cell maintenance, organismal growth, and mammary gland development Janus kinases (JAKs) were identified through sequence comparisons as a unique class of tyrosine kinases that contain both a catalytic domain and a second kinase-like domain that serves an autoregulatory function. They were functionally linked to STATs and interferon signaling in powerful somatic cell genetic screens. The JAK/STAT cascade is among the simplest of the conserved metazoan signaling pathways. The binding of extracellular ligand leads to pathway activation via changes to the receptors that permit the intracellular JAKs associated with them to phosphorylate one another. Trans-phosphorylated JAKs then phosphorylate downstream substrates, including both the receptor and the STATs. Activated STATs enter the nucleus and bind as dimers or as more complex oligomers to specific enhancer sequences in target genes, thus regulating their transcription -Binding of erythropoietin (EPO) causes dimerization of the EPO receptor, which allows JAK, a soluble Tyrkinase, to bind to the internal domain of the receptor and phosphorylate it on several Tyr residues. (a) In one signaling pathway, the SH2 domain of the STAT protein STAT5 binds to P-Tyr residues on the receptor, bringing it into proximity with JAK. Following phosphorylation of STAT5 by JAK, two STAT5 molecules dimerize, each binding the other's P-Tyr residue, thus exposing a nuclear localization sequence (NLS) that targets the dimer for transport into the nucleus. In the nucleus, STAT5 turns on the expression of EPO-controlled genes. (b) In a second signaling pathway, following EPO binding and autophosphorylation of JAK, the adaptor protein Grb2 binds P-Tyr in JAK and triggers the MAPK cascade, as in the insulin system
Describe the structure and function of calmodulin. What happens when it binds Ca2+? How the troponin C similar to calmodulin?
-Changes in intracellular [Ca2+] are detected by Ca2+ binding proteins that regulate a variety of Ca2+ dependent enzymes. -Calmodulin (CaM) is an acidic protein with four high-affinity Ca2+ binding sites. When intracellular [Ca2+] rises to about 106 M (1 μM ), the binding of Ca2+ to calmodulin drives a conformational change in the protein. Associates with a variety of proteins and, in its Ca2+ bound state, modulates their activities. This family shares a characteristic Ca2+ binding structure, the EF hand (helix-loop-helix motif) -An integral subunit of a family of enzymes, the Ca2+/calmodulin-dependent protein kinases (CaM kinases I-IV). When intracellular [Ca2+] increases in response to some stimulus, calmodulin binds Ca2+, undergoes a change in conformation, and activates the CaM kinase. The kinase then phosphorylates a number of target enzymes, regulating their activities. Calmodulin is also a regulatory subunit of phosphorylase b kinase of muscle, which is activated by Ca2+. Thus Ca2+ triggers ATP-requiring muscle contractions while also activating glycogen breakdown, providing fuel for ATP synthesis. Many other enzymes are also modulated by Ca2+ through calmodulin
Explain the bind and function of β-arrestin.
-Desensitization damps a signal response while the signal is still persisting. Desensitization of the beta-adrenergic receptor in the continued presence of epinephrine is mediated by a protein kinase that phosphorylates the receptor on the interacellular domain that normally interacts with Gs. -When the receptor is occupied by epinephrine, Beta- adrenergic receptor kinase (BetaARK) phosphorylates several Ser residues near the carboxyl terminus of the receptor which is usually on the cytoplasmic side of the plasma membrane. BARK is drawn to the plasma membrane by its associate with the GsBy subunits and is thus positioned to phosphorylate the receptor. Receptor phosphorylation creates a binding site for the protein B-arrestin. and the binding of beta arrestin prevents further interaction between the receptor and the G protein. -The binding of beta-arrestin also facilitates receptor sequestration, the removal of receptor molecules from the plasma membrane by endocytosis into small intracellular vesicles. The arrestin-receptor complex recruits two proteins involved in vesicle formation, the AP-2 complex and clathrin, which then initiate membrane invagination leading to the formation of the endosomes containing the adrenergic receptor. Here now the receptors are inaccessible to epinephrine and therefore inactive.
Describe the general structure of G protein receptor (GPRC). Describe the relation ship between Ga-GDP and GbGg. How are these tethered to the plasma membrane? How do they interact with the GPRC?
-GPCR: A GPCR is a plasma membrane receptor with seven transmembrane helical segments. It is associated with a G-protein that cycles between active, GTP bound form, and inactive or GDP- bound form. There is also an effector enzyme or ion channel that is regulated by the activated G protein.GPCRs are heterotrimeric, meaning they have three different subunits: an alpha subunit, a beta subunit, and a gamma subunit. Two of these subunits — alpha and gamma — are attached to the plasma membrane by lipid anchors -Three essential components define signal transduction through GPCRs: a plasma membrane receptor with seven transmembrane helical segments, a G protein that cycles between active (GTP-bound) and inactive (GDP-bound) forms, and an effector enzyme (or ion channel) in the plasma membrane that is regulated by the activated G protein. 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. A G protein alpha subunit binds either GTP or GDP depending on whether the protein is active (GTP) or inactive (GDP). In the absence of a signal, GDP attaches to the alpha subunit, and the entire G protein-GDP complex binds to a nearby GPCR. This arrangement persists until a signaling molecule joins with the GPCR. At this point, a change in the conformation of the GPCR activates the G protein, and GTP physically replaces the GDP bound to the alpha subunit. As a result, the G protein subunits dissociate into two parts: the GTP-bound alpha subunit and a beta-gamma dimer. Both parts remain anchored to the plasma membrane, but they are no longer bound to the GPCR, so they can now diffuse laterally to interact with other membrane proteins. G proteins remain active as long as their alpha subunits are joined with GTP. However, when this GTP is hydrolyzed back to GDP, the subunits once again assume the form of an inactive heterotrimer, and the entire G protein reassociates with the now-inactive GPCR. In this way, G proteins work like a switch — turned on or off by signal-receptor interactions on the cell's surface.
What is the function of retinoblastoma protein?
-It is a very important CDK substrate. When DNA damage is detected, the pRb protein participates in a mechanism that arrests cell division in G1. It functions in most cell types to regulate cell division in a response to various stimuli. The mechanism described below gives the cell time to repair its damaged DNA before it enters into the S phase, which ultimately avoids the potentially disastrous transfer of a defective genome to one of both daughter cells. -Mechanism: When pRb is unphosphorylated, it binds to transcription factor E2F. When it is bound to E2F, E2F is unable to promote transcription of a group of genes that are necessary for DNA synthesis. Therefore, the cell cycle cannot proceed from the G1 phase to the S phase. The pRB-E2F blocking mechanism is relieved when pRB is phosphorylated by cyclin E-CDK2, and this phosphorylation happens in response to a signal for cell division to proceed. When the protein kinases ATM and ATR detect DNA damage, they activate p53 to serve as a transcription factor to stimulate the synthesis of p21. p21 inhibits the protein kinase activity of cyclin E-CDK2; therefore, in the presence of p21, pRB remains unphosphorylated and bound to E2F, blocking the activity of this transcription factor, and ultimately arresting the cell cycle in G1. -This gives the cell time to repair its DNA before entering the S phase, thereby avoiding the potentially disastrous transfer of a defective genome to one or both daughter cells. When the damage is too severe to allow effective repair, this same machinery triggers a process, apoptosis, that leads to the death of the cell, preventing the possible development of a cancer.
Describe the MAP kinase cascade. What is the function of scaffold protein?
-MAPK cascades is a cascade of reactions that amplifies the initial signal by many orders of magnitudes. It mediates signaling initiated by a variety of growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). Regulation of gene expression by insulin occurs through a MAP kinase cascade. -1. Insulin receptor binds insulin and undergoes autophosphorylation on its carboxyl-terminal Tyr residues. 2. Insulin receptor phosphorylates IRS-1 on its Tyr residues 3. SH2 domain of Grb2 binds to P-Tyr of IRS-1. Sos binds to Grb2, then to Ras, causing GDP release and GTP binding to Ras. 4. Activated Ras binds and activates Raf-1. 5. Raf-1 phosphorylates MEK on two Ser residues, activating it. MEK phosphorylates ERK on a Thr and Tyr residue, activating it. 6. ERK moves into the nucleus and phosphorylates nuclear transcription factors such as Elk1, activating them 7. Phosphorylated Elk1 joins SRF to stimulate the transcription and translation of a set of genes needed for cell division -Scaffold proteins act in at least four ways: tethering signaling components, localizing these components to specific areas of the cell, regulating signal transduction by coordinating positive and negative feedback signals, and insulating correct signaling proteins from competing proteins.
Describe IP3, Ca2+ and DAG as second messages. How do they arise? Describe the roles of PLC and PKC.
-Many different GPCR receptors that activate different G proteins that can activate other enzymes other than adenylate cyclase. For example, an activated G protein can activate PLC, which is Phospholipase C. PLC can act on phospholipids and hydrolyze them. These are lipids that are anchored to the membrane and contain a phosphate group and sugar group. PLC with cut between the lipids and the phoshphate sugar to produce DAG (diacyl glycerol) and IP3. IP3 (inositol 1,4,5-triphosphate (IP3) is a sugar covered with three phosphate molecules. DAG is glycerol with one empty hydroxyl group and two lipids anchored into the membrane. DAG is able to activate another kinase, PKC. PKC and PLC are both enzymes that add phosphate onto target proteins, but have different targets. IP3 is a second messenger that can cause the release of calcium from intercellular storages from the ER. IP3 binds to a calcium channel on the ER and allows the release of Calcium into the cytoplasm. This causes a higher increase in the Ca2+ concentration in the cytoplasm of the cell all due to the binding a signal molecule to the GPCR. Ca2+ can also stimulate PKC, so PKC is being activated by both DAG and PKC. -Ca2+ has many important roles as an intracellular messenger. The release of a large amount of free Ca2+ can trigger a fertilized egg to develop, skeletal muscle cells to contract, secretion by secretory cells and interactions with Ca2+ -responsive proteins like calmodulin. To maintain low concentrations of free Ca2+ in the cytosol, cells use membrane pumps like calcium ATPase found in the membranes of sarcoplasmic reticulum of skeletal muscle. These pumps are needed to provide the steep electrochemical gradient that allows Ca2+ to rush into the cytosol when a stimulus signal opens the Ca2+ channels in the membrane. The pumps are also necessary to actively pump the Ca2+ back out of the cytoplasm and return the cell to its pre-signal state
How does the binding of insulin cause the presence of GLUT 4 on cell membranes?
-PI-3K binds IRS-1 through the former's SH2 domain. Thus activated, PI-3K converts the membrane lipid PIP2 to PIP3. When bound to PIP3, PKB is phosphorylated and activated by yet another protein kinase PDK1. The activated PKB then phosphorylates Ser or Thr residues on its target proteins, one of which is glycogen synthase kinase 3 (GSK3). In its acive, nonphos. Form, GSK2 phosphorylates glycogen synthase, inactivating it and contributing to the slowing of glucogen synthesis When phosphorylated by PKB, GSK3 is inactivated. By thus preventing inactivation of glycogen synthase in liver and muscle, the cascade of protein phosphorylations initiated by insulin stims glycogen synthesis (12-8). In muscle, PKB triggers the movement of glucose transporters (GLUT4) from internal vesicles to the plasma membrane, stimulating glucose uptake from the blood.
cRas is a G protein; (a) How is it activated? (b) Compare and contrast Ras with Ga _protein. For each of these, what acts as GTP exchange factor?
-Ras is the prototype of a family of small G proteins that mediate a wide variety of signal transductions. Like the trimeric G protein that functions with the B-adrengenic system, Ras can exist in either the GTP-bound (active) or GDP-bound (inactive) conformation, but Ras acts as a monomer. When GTP binds, Ras can activate a protein kinase Raf-1, the first of three protein kinases, Raf-1, MEK, ERK, that form a cascade in which each kinase activates the next by phosphorylation. Ras is a G protein, or a guanosine-nucleotide-binding protein. Specifically, it is a single-subunit small GTPase, which is related in structure to the Gα subunit of heterotrimeric G proteins (large GTPases). G proteins function as binary signaling switches with "on" and "off" states. In the "off" state it is bound to the nucleotide guanosine diphosphate (GDP), while in the "on" state, Ras is bound to guanosine triphosphate (GTP), which has an extra phosphate group as compared to GDP. This extra phosphate holds the two switch regions in a "loaded-spring" configuration (specifically the Thr-35 and Gly-60). When released, the switch regions relax which causes a conformational change into the inactive state. Hence, activation and deactivation of Ras and other small G proteins are controlled by cycling between the active GTP-bound and inactive GDP-bound forms.
There are approximately 21 structurally related RTK's. Describe the common features of this family. What make the Insulin RTK unique?
-Receptor tyrosine kinases (RTKs), a large family of plasma membrane receptors with intrinsic protein kinase activity, transduce extracellular signals by a mechanism fundamentally different from that of GPCRs. These receptor proteins have a ligand-binding domain on the extracellular surface of the plasma membrane and an enzyme active site on the cytosolic side, with the two domains connected by a single transmembrane segment. Usually, the receptor enzyme is a protein kinase that phosphorylates Tyr residues in their specific target proteins. -The insulin receptor is the prototype for a number of receptor enzymes with a similar structure and receptor Tyr kinase activity. The receptors for epidermal growth factor and platelet-derived growth factor, for example, have structural and sequence similarities to the insulin receptor, and both have a protein Tyr kinase activity that phosphorylates IRS-1. Many of these receptors dimerize after binding ligand; the insulin receptor is already a dimer before insulin binds. The binding of adaptor proteins such as Grb2 to P -Tyr residues is a common mechanism for promoting protein-protein interactions. (unbound to insulin as a homo dimer) -Proteins that bind to it are specific scaffolding and adapter proteins
What are the roles of the SH-2 and SH-3 domains? What are the functions of Grb2 and Sos?
-Several signaling proteins contain SH2 domains, all of which bind P-Tyr residues in a protein partner. -Several signaling proteins contain SH2 domains, all of which bind P-Tyr residues in a protein partner. One of these target proteins (Fig. 12-6, step 2 ) is insulin receptor substrate-1 (IRS-1). Once phosphorylated on its Tyr residues, IRS-1 becomes the point of nucleation for a complex of proteins (step 3) that carry the message from the insulin receptor to end targets in the cytosol and nucleus, through a long series of intermediate proteins. First, a P -Tyr residue in IRS-1 is bound by the SH2 domain of the protein Grb2. (SH2 is an abbreviation of Src homology 2 ; the sequences of SH2 domains are similar to a domain in another protein Tyr kinase, Src) Grb2 also contains a second protein-binding domain, SH3, that binds to regions rich in Pro residues. Grb2 binds to a proline-rich region of Sos, recruiting Sos to the growing receptor complex. When bound to Grb2, Sos catalyzes the replacement of bound GDP by GTP on Ras, one of a family of guanosine nucleotide-binding proteins (G proteins) that mediate a wide variety of signal transductions (Section 12.4). When GTP is bound, Ras can activate a protein kinase, Raf-1 (step 4 ), the first of three protein kinases—Raf-1, MEK, and ERK—that form a cascade in which each kinase activates the next by phosphorylation (step 5 ). The protein kinase ERK is activated by phosphorylation of both a Thr and a Tyr residue. When activated, it mediates some of the biological effects of insulin by entering the nucleus and phosphorylating proteins such as Elk1, which modulates the transcription of about 100 insulin-regulated genes (step 6 ). Grb2 is an adapter protein with no intrinsic enzymatic activity. Its function is to bring together two proteins that must interact to enable signal transduction. In addition to its SH2 (P-Tyr binding) domain, Grb2 also contains a second protein-binding domains, SH3, that binds to a proline-rich region of Sos, recruiting Sos to the growing receptor complex. When bound to Grb2, Sos acts as a guanosine nucleotide-exchange factor (GEF), catalyzing the replacement of bound GDP with GTP on Ras, a G protein.
Explain specificity of ligand.
-Specificity of a ligand means that only that specific signal molecule fits the binding site on its complementary receptor and other signals do not fit Mediated through the same kinds of weak or non covalent forces that mediate enzyme-substrate and antigen-antibody interactions Specificity is achieved at higher levels in multicellular organisms because receptors for a given signal or the intracellular targets of a given signal pathway are present in only specific cell types. This means that other cell types do not even have the receptors and cannot read the signal. For example, thyrotropin-releasing hormone triggers response in the cells of the anterior pituitary but not in the hepatocytes which lack the receptors for this hormone.
Describe switch 1 and 2 and the P loops role in GTP binding and hydrolysis.
-Studies have revealed the location of the nucleotide-binding pocket and the interface between Gα and Gβγ subunits. The nucleotide-binding pocket is located between Ras-like domain and α-helical domain of Gα subunit surrounded by four flexible regions (p-loop , switch I, switch II, and switch III). Ras-like domain hydrolyze GTP and provide binding sites for Gβ subunit. The N-terminus of Gα subunit is reported to be critical for the structure and function of Gα subunit and is myristolylated or palmitoylated suggesting the role of this region in the attachment to the plasma membrane. In the GTP-bound conformation, the G protein exposes previously buried regions, called switch I and switch II that interact with proteins downstream in the signaling pathway, until the G protein inactivates itself by hydrolyzing its bound GTP to GDP. The critical determinant of G-protein conformation is the y phosphate of GTP, which interacts with a region called the P-loop (phosphate binding). In Ras, the y phosphate binding of GTP binds to a Lys residue in the P loop and to two critical residues, Thr35 in switch I and Gly60 in switch II, that hydrogen bond with the oxygens of the y phosphate of GTP. These hydrogen bonds act like 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. Ala146 hydrogen bonds to the guanine oxygen, allowing GTP, but not ATP to bind.
How does phosphorylation affect the activity of CDKs?
-The activity of a CDK is affected by phosphorylation and dephosphorylation of two critical residues in the protein. When the Tyr near the amino terminus is phosphorylated, the CDK2 is made inactive. It is inactive because the phosphorylated Tyr residue is in the ATP-binding site of the kinase, and the negatively charged phosphate group blocks the entry of ATP. A specific phosphatase dephosphorylates this P-Tyr residue, permitting the binding of ATP. Phosphorylation of Thr160 in the "T loop" of CDK, catalyzed by the CDK-activating kinase, forces the T loop out of the substrate-binding cleft, permitting substrate binding and catalytic activity. -one example of triggering this control mech: single strand breaks in DNA, arrest in cell cyle in G2. Cell will not divide until DNA repaired.
What are the roles of cyclins and CDKs in the advancement through the ell cycle?
-The kinases are heterodimers with a regulatory subunit, cyclin, and a catalytic subunit, cyclin-dependent protein kinase(CDK). In the absence of cyclin, the catalytic subunit is virtually inactive. When cyclin binds, the catalytic site opens up, a residue essential to catalysis becomes accessible, and the activity of the catalytic subunit increases 10,000 fold. -In a population of animal cells undergoing synchronous division, some CDK activities show striking oscillation. The precisely timed activation and inactivation of a series of CDKs produce signals serving as a master clock that orchestrates the events in normal cell division and ensures that one stage is completed before the next begins. a. Without cyclin, CDK2 folds so that one segment, the T loop, obstructs the binding site for protein substrates and thus inhibits protein kinase activity. The binding site for ATP is also near the T loop. b. When cyclin binds, it forces conformational changes that move the T loop away from the active site and reorient an amino-terminal helix, bringing a residue critical to catalysis (Glu51) into the active site c. Phosphorylation of a Thr residue in the T loop produces a negatively charged residue that is stabilized by interaction with three Arg residues, holding CDK in its active conformation.
How is the interaction of IRS different from that of Grb2 and Sos?
-The signaling pathway from insulin branches at IRS-1. Grb2 is not the only protein that associates with phosphorylated IRS-1. The enzyme phosphoinositide 3-kinase (PI3K) binds IRS-1 through PI3K's SH2 domain. Thus activated, PI3K converts the membrane lipid phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate. -?
How are cell stimulated to enter the cell cycle?
-The timing of the cell cycle is controlled by a family of protein kinases with activities that change in response to cellular signals. The kinases are heterodimers with a regulatory subunit, cyclin, and a catalytic subunit, cyclin-dependent protein kinase(CDK). In the absence of cyclin, the catalytic subunit is virtually inactive. By phosphorylating specific protein at precisely timed intervals, these protein kinases orchestrate the metabolic activities of the cell to produce orderly cell division.
How does the GPRC act as the guanine exchange factor (GEF) for Ga?
-When a ligand binds to the GPCR, it causes a conformational change in the GPCR, which allows it to act as a guanine nucleotide exchange factor (GEF). -The GPCR can then activate an associated G protein by exchanging its bound GDP for a GTP. -G protein's α subunit, together with the bound GTP, can then dissociate from the β and γ subunits to further affect intracellular signaling proteins or target functional proteins directly depending on the α subunit type.
Describe the steps in the molecular circuit of signal transduction. How does cooperativity aid in the transmission of signal from low concentrations of ligand?
-general features of signal transduction: a signal interacts with a receptor; the activated receptor interacts with cellular machinery, producing a second signal or a change in the activity of a cellular protein; the metabolic activity (broadly defined to include metabolism of RNA, DNA, and protein) of the target cell undergoes a change; and finally, the transduction event ends and the cell returns to its pre stimulus state -3 factors account for the extraordinary sensitivity of signal transducers: High affinity of receptors for signal molecules Cooperativity (often but not always) in the ligand-receptor interaction Amplification of the signal by enzyme cascades -Specificity: achieved by precise molecular complementarity between the signal and receptor molecules, mediated by the same kinds of weak (noncovalent) forces that mediate enzyme-substrate and antigen-antibody interactions. -When a ligand binds, it can increase the binding affinity for other ligands, thus cooperativity aid in the transmission of signal from low concentrations of ligand (like coopertivity of O in hemoglobin) (Cooperativity in receptor-ligand interactions results in large changes in receptor activation with small changes in ligand concentration.)
Describe the sequence of events when a ligand binds to a GPRC.
1. Ligand binds to receptor (example of a ligand could be epinephrine and receptor is adrenergic receptor) 2. G protein is stimulated by activated receptor and exchanged GDP for GTP 3. G protein dissociated from the occupied receptor and binds to nearby effector enzyme This activated effector enzyme (example could be adenylyl cyclase) 4. The activated enzyme then generates a second messenger that affects downstream targets. (could be cAMP) 6. Downstream targets are activated (example cAMP activated PKA) 7. In the case of PKA (protein kinase A) it phosphorylates other cellular proteins that is the effect of the ligand that bound to original receptor Details: -The binding of epinephrine to a site on the receptor deep within the membrane promotes a conformational change in the receptor's intracellular domain that affects its interaction with the second protein in the signal-transduction pathway, a heterotrimeric GTP-binding stimulatory G protein, or Gs, on the cytosolic side of the plasma membrane. -When the nucleotide-binding sire of Gs (on the alpha subunit) is occupied by GTP, Gs is active and can activate adenylyl cyclase; with GDP bound to the site, Gs is inactive. Binding of epinephrine enables the receptor to catalyze displacement of bound GDP by GTP, converting Gs to its active form. (2). As this occurs, B and gamma subunits of Gs dissociate from the a subunit, and Gsa, with its bound GTP, moves in the plane of the membrane from the receptor to a nearby molecule of adenylyl cyclase (3). -The Gsa is held to the membrane by a covalently attached palmitoyl group. The association of active Gsa with adenylyl cyclase stimulates the cyclase to catalyze cAMP synthesis (4), raising the cytosolic [cAMP]. This stimulation by Gsa is self-limiting; Gsa is a GTPase that turns itself off by converting its bound GTP to GDP. --cAMP does not affect phosphorylase b kinase directly. Rather, cAMP dependent protein kinase (kinase A or PKA), which is allosterically activated by cAMP (5), catalyzes the phosphorylation of inactive phosphorylase b kinase to yield the active form. The inactive form of PKA contains 2 catalytic subunits © and 2 regulatory subunits ®. The tetrameric R2C2 complex is catalytically inactive, because an autoinhibitory domain of each R subunit occupies the substrate-binding site of each C subunit. When cAMP binds to two sites on each R subunit, the R subunits undergo a conformational change and the R2C2 complex dissociates to yield two free catalytically active C subunits. -(6)PKA regulates a number of enzymes. Although the proteins regulated by cAMP-dependent phosphorylation have diverse functions, they share a region of seq similarity around the Ser or Thr residue that undergoes phosphorylation, a seq that marks them for regulation by PKA. -(7): cyclic AMP, second messenger, is short-lived. It is quickly degraded by cyclic nucleotide phosphodiesterase to 5'-AMP, which is not active as a second messenger.
Ga _has GTPase activity. The hydrolysis of GTP to GDP inactivates Ga. How would a mutation in Ga _affect the cascade? (a) Increased GTPase catalytic turnover rate? (b) Decreased GTPase catalytic rare? (c) Decreased affinity for GDP?
a) inactivates Ga and lowers the cellular response through the hydrolysis of GTP. b) increasing the response c) GTP can easily displace it; activated and increase response