Presentation on theme: "Figure 15-30 1234567 G-protein-coupled receptors (GPCRs) G-protein-linked receptors mediate cellular responses to a wide variety of signaling molecules."— Presentation transcript:
Figure 15-30 1234567 G-protein-coupled receptors (GPCRs) G-protein-linked receptors mediate cellular responses to a wide variety of signaling molecules. All possess a similar architecture and consist of a single polypeptide chain that traverses the bilayer seven times.
These receptors active their targets indirectly, via a G protein intermediate. G protein-mediated signaling
Figure 15-31 Structure of an inactive trimeric G protein Trimeric GTP-binding proteins (G proteins) functionally couple GPCR receptors to their target proteins. The G protein consists of three subunits: , and . The subunit binds guanine nucleotides and is active when bound to GTP.
G-protein-linked Receptors * When GPCRs bind ligand they undergo a conformational change that allows them to interact with the G protein. * This interaction causes the G protein to eject the bound GDP and replace it with GTP. * This exchange causes the trimer to dissociate into two activated components: an subunit bound to GTP and a complex.
* Receptor activation generally results in the production of intracellular (or second) messengers that in turn pass the signal on to additional cellular proteins. * Two of the most common second messengers used in these signaling pathways are cAMP and Ca 2+. activated complex activated enzyme activated subunit of G protein Many intracellular messenger molecules diffuse widely to act on target proteins in various parts of the cell
G proteins & Adenylyl Cyclase * Ligand binding by some G-protein-linked receptors results in the activation of adenylyl cyclase. * The subunit of the stimulatory G protein (G S ) carries the signal from these receptors to adenylyl cyclase. * Binding to adenylyl cyclase stimulates the intrinsic GTPase activity of the S subunit. The hydrolysis of GTP inactivates S and hence adenylyl cyclase as well. * The effects of cAMP are primarily mediated by the cAMP- dependent protein kinase (PKA). * PKA activates the transcription of specific genes by phosphorylating and activating regulatory proteins, such as CREB. * CREB binds to the cAMP response element (CRE) in the promoter region of genes and stimulates their transcription.
Figure 15-36 Increased [cAMP] influences gene expression in the nucleus
Substrate Substrate- P Kinase (PKA) Phosphatase * The effects of PKA are reversed by protein phosphatases that remove the phosphates put on by PKA. * The activity of proteins regulated by phosphorylation depends upon the relative levels of the protein kinase and phosphatase present in the cell. Protein phosphorylation is a reversible modification
Extracellular signals can be greatly amplified by the use of intracellular mediators and enzymatic cascades. This is demonstrated here with the adenylyl cyclase-PKA pathway.
Biochemical Engineering: Identifying Protein Kinase Inhibitors Goal: Design specific inhibitors for protein kinases Problem: Most protein kinase inhibitors act are structurally similar to ATP and compete for binding in the active site with this nucleotide. Since the active site structure has been well conserved through evolution, it can be difficult to find drugs that are specific for one particular protein kinase. Kevan Shokat Solution: Take advantage of known structural information and use a biochemical engineering approach to inhibitor design.
Analog-sensitive Protein Kinases Dr. Shokat noted that the ATP binding pocket of most PKs had an amino acid residue with a large bulky group at the back of the pocket. This residue is known as the “gatekeeper.” He reasoned that ATP-competitive inhibitors that had a large side group present at the position that abuts this gatekeeper residue would not fit into the wild-type enzyme pocket. However, changing this position to a residue with a smaller side chain (like glycine) would accommodate the new inhibitor. Kinase to be targeted Fully functional kinase with altered gatekeeper Highly specific inhibitor binds targeted enzyme to inhibit kinase activity “Holed” Enzyme Met Gly Gatekeeper M 120 ATP
Result: A potent method for inhibiting a given protein kinase Wild-type enzymeMutated enzyme
G proteins & Inositol Phospholipid Signaling * Many GPCRs exert their effects via G proteins that activate the plasma membrane-bound enzyme, phospholipase C- (PLC- ). * Activated PLC- cleaves PIP 2 to generate two products: inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DIG). * IP 3 is a water-soluble signaling molecule that triggers the opening of IP 3 -gated Ca 2+ -release channels in the ER membrane. * The opening of these channels results in the release of the Ca 2+ stored in the ER lumen. * DIG remains in the plasma membrane and contributes, along with Ca 2+, to the activation of protein kinase C (PKC). * Several mechanisms attenuate signaling through this pathway including the phosphorylation/dephosphorylation of IP 3, and the pumping of the cytosolic Ca 2+ to the exterior of the cell.
Many GPCRs exert their effects via G proteins that activate the plasma membrane-bound enzyme, phospholipase C- (PLC- ) G proteins & Inositol Phospholipid Signaling
Activated PLC- cleaves PIP 2 to generate two products: inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DIG) G proteins & Inositol Phospholipid Signaling
Figure 15-37 Different phosphorylated forms of phosphatidylinositol
The downstream effects of the two signaling products of PIP 2 cleavage, IP 3 and DIG
Figure 15-41 Maintaining a low [Ca 2+ ] in the cytoplasm Plasma membrane pumps actively transport Ca 2+ to the exterior of the cell
Figure 15-41 Maintaining a low [Ca 2+ ] in the cytoplasm Ca 2+ is also pumped into both the ER and mitochondria, and various molecules in the cytoplasm bind to free Ca 2+.
Ca 2+ is a ubiquitous intracellular mediator * Many extracellular signals trigger an increase in cytosolic Ca 2+ concentration. The Ca 2+ enters the cytoplasm from either the outside of the cell or from the lumen of the ER. * A variety of Ca 2+ -binding proteins help to relay the cytosolic Ca 2+ signal. * The most important of these is calmodulin, an abundant protein that acts as an multipurpose Ca 2+ -receptor in all eukaryotic cells. * The Ca 2+ /calmodulin complex binds to a number of target proteins including the Ca 2+ /calmodulin-dependent protein kinases (CaM-kinases). * One of the best-studied is CaM-kinase II that exhibits a molecular memory as a result of a specific autophosphorylation reaction.
Figure 15-43 Calmodulin is an important Ca 2+ -binding protein in the cytoplasm Substrate binding often results in substantial structural changes in the Ca 2+ /calmodulin complex
Figure 15-44 The stepwise activation of CaM-kinase II (1) (2)
Protein kinase substrate identification ➠ Protein kinases function by phosphorylating particular targets and altering their biological activity ➠ To understand the function of any given protein kinase, we have to identify its particular substrates. ➠ A number of interesting strategies have been developed in recent years to assist this identification process.
Mass Spectrometry: Biological Applications Separates molecules on the basis of their mass. Can identify peptides that differ by 80 Da, the size of a phosphate group.
Figure 15-52 Ligand Binding Kinase Domain Receptor tyrosine kinase subfamilies The receptors for most growth factors are transmembrane tyrosine-specific protein kinases. The intracellular kinase domain is activated by ligand binding.
Figure 15-53a Activation of a receptor tyrosine kinase Ligand binding triggers receptor dimerization and the cross- phosphorylation of cytoplasmic domains (autophosphorylation). The autophosphorylated tyrosines act as high affinity binding sites for a variety of intracellular signaling proteins that contain an SH2 domain.
Activation of a receptor tyrosine kinase Autophosphorylation contributes to activation process in two ways: 1. Phosphorylation of tyrosine residues increases kinase activity; 2. Phosphorylated tyrosines serve as docking sites for other signaling molecules.
Figure 15-54 Assembly of a transient intracellular signaling complex Tyrosine autophosphorylation of the receptors serves to trigger the transient assembly of an intra- cellular signaling complex.
Figure 15-55a Binding of SH2-containing proteins to activated PDGF receptors
Ras Proteins * Monomeric GTP-binding proteins that link receptor tyrosine kinases to other downstream signaling molecules. * Originally identified as the hyperactive products of mutant ras genes associated with mammalian cancers. * The Ras proteins oscillate between an active GTP-bound form and an inactive GDP-bound form. * Ras activity is modulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). * The Ras proteins activate a phosphorylation cascade that involves a MAP kinase, ERK (extracellular signal-regulated kinase). * Activation of such protein kinases often results in both a transient immediate response and a prolonged, delayed set of responses.
Figure 15-19 GAP = GTPase-activating protein GEF =Guanine nucleotide exchange factor The regulation of Ras activity Ras
PI 3-kinase Signaling * The PI 3-kinase enzyme binds to the intracellular domains of activated RTK molecules. * This kinase primarily phosphorylates inositol phospholipids at the 3’ position of the inositol ring rather than proteins. * One of these products, PI(3,4,5)P 3, serves as a docking site for various signaling proteins. * Some of these latter proteins contain a pleckstrin homology (PH) domain that mediates the interaction with PI(3,4,5)P 3. * The protein kinase, Akt, is a PH domain-containing protein that is part of a signaling pathway generally important for promoting cell growth and survival.
Figure 15-63 Generation of PI-based docking sites by PI 3-kinase Figure 13-10
Interconversion possibilities that exist between the different forms of PI
Figure 15-63 Generation of PI-based docking sites by PI 3-kinase Figure 13-10 PI(3)PPI(4,5)P 2 Phosphoinositide head groups are recognized by protein domains that discriminate between the differently-phosphorylated forms.
PH domain protein cytosol PI(3,4,5)P 3 docking site plasma membrane Pleckstrin homology (PH) domains can mediate binding to PI(3,4,5)P 3
1. Production of PI(3,4,5)P 3 The PI3K-Akt signaling pathway is the major pathway activated by the hormone insulin and the insulin-like growth factors (IGFs). Members of the insulin-like growth factor (IGF) family of signal proteins stimulate many animal cells to survive and grow. These IGFs bind to specific RTKs that activate PI3K to produce PI(3,4,5)P 3.
mTORC2 2. Recruitment & activation of Akt Note that Akt requires two phosphorylation events for activation. PDK1 = Phosphoinositol-Dependent Kinase 1
3. Phosphorylation of Bad & inhibition of apoptosis
Target Cell Adaptation * Following prolonged exposure to a stimulus, target cells can undergo a process of adaptation or desensitization. * As a result, cells can detect the same percent change in a signal over a wide range of stimulus intensities. * Slow adaptation is achieved by the process of receptor down-regulation. * Rapid adaptation often involves phosphorylation of the cytoplasmic tail of the cell-surface receptor. * Target cell adaptation may arise as a consequence of modulating later steps in the signaling cascade.