G-protein-coupled receptors (GPCRs) 1 2 3 4 5 6 7 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. Figure 15-30

G protein-mediated signaling These receptors active their targets indirectly, via a G protein intermediate.

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. Figure 15-31

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.

Figure 15-32

activated a subunit of G protein activated bg complex activated enzyme activated a subunit of G protein Many intracellular messenger molecules diffuse widely to act on target proteins in various parts of the cell 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 Ca2+.

Figure 15-34 Molecular Biology of the Cell (© Garland Science 2008)

Table 15-1 Molecular Biology of the Cell (© Garland Science 2008)

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 (GS) 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.

Intracellular effects of increasing cAMP levels Figure 15-36 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)

The cAMP-dependent protein kinase (PKA) is the primary effector of cAMP Figure 15-35 Molecular Biology of the Cell (© Garland Science 2008)

Increased [cAMP] influences gene expression in the nucleus Figure 15-36

Protein phosphorylation is a reversible modification Substrate Kinase (PKA) Phosphatase Substrate- P 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.

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 Gatekeeper M120 ATP 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

Result: A potent method for inhibiting a given protein kinase Mutated enzyme Wild-type enzyme

Table 15-3 Molecular Biology of the Cell (© Garland Science 2008)

Table 15-2 Molecular Biology of the Cell (© Garland Science 2008)

G proteins & Inositol Phospholipid Signaling Many GPCRs exert their effects via G proteins that activate the plasma membrane-bound enzyme, phospholipase C-b (PLC-b). Activated PLC-b cleaves PIP2 to generate two products: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DIG). IP3 is a water-soluble signaling molecule that triggers the opening of IP3-gated Ca2+-release channels in the ER membrane. The opening of these channels results in the release of the Ca2+ stored in the ER lumen. DIG remains in the plasma membrane and contributes, along with Ca2+, to the activation of protein kinase C (PKC). Several mechanisms attenuate signaling through this pathway including the phosphorylation/dephosphorylation of IP3, and the pumping of the cytosolic Ca2+ to the exterior of the cell.

G proteins & Inositol Phospholipid Signaling Many GPCRs exert their effects via G proteins that activate the plasma membrane-bound enzyme, phospholipase C-b (PLC-b)

G proteins & Inositol Phospholipid Signaling Activated PLC-b cleaves PIP2 to generate two products: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DIG)

Different phosphorylated forms of phosphatidylinositol Figure 15-37

Hydrolysis of PI(4,5)P2 by phospholipase C-b Figure 15-38 Molecular Biology of the Cell (© Garland Science 2008)

GPCRs increase cytosolic Ca2+ and activate PKC Figure 15-39 Molecular Biology of the Cell (© Garland Science 2008)

The downstream effects of the two signaling products of PIP2 cleavage, IP3 and DIG

Maintaining a low [Ca2+] in the cytoplasm Plasma membrane pumps actively transport Ca2+ to the exterior of the cell Figure 15-41

Maintaining a low [Ca2+] in the cytoplasm Ca2+ is also pumped into both the ER and mitochondria, and various molecules in the cytoplasm bind to free Ca2+. Figure 15-41

Ca2+ is a ubiquitous intracellular mediator Many extracellular signals trigger an increase in cytosolic Ca2+ concentration. The Ca2+ enters the cytoplasm from either the outside of the cell or from the lumen of the ER. A variety of Ca2+-binding proteins help to relay the cytosolic Ca2+ signal. The most important of these is calmodulin, an abundant protein that acts as an multipurpose Ca2+-receptor in all eukaryotic cells. The Ca2+/calmodulin complex binds to a number of target proteins including the Ca2+/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.

Calmodulin is an important Ca2+-binding protein in the cytoplasm Substrate binding often results in substantial structural changes in the Ca2+/calmodulin complex Figure 15-43

The stepwise activation of CaM-kinase II (1) (2) Figure 15-44

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.

Signaling with enzyme-coupled receptors Figure 15-16c Molecular Biology of the Cell (© Garland Science 2008)

Receptor tyrosine kinase subfamilies Ligand Binding Kinase Domain The receptors for most growth factors are transmembrane tyrosine-specific protein kinases. The intracellular kinase domain is activated by ligand binding. Figure 15-52

Table 15-4 Molecular Biology of the Cell (© Garland Science 2008)

Activation of a receptor tyrosine kinase Figure 15-53a 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.

PDGF Signaling

HGH binds as a monomer to its dimeric receptor The three-dimensional structure of human growth hormone bound to its receptor HGH binds as a monomer to its dimeric receptor Figure 15-67 Molecular Biology of the Cell (© Garland Science 2008)

Activation of a receptor tyrosine kinase Autophosphorylation contributes to activation process in two ways: Phosphorylation of tyrosine residues increases kinase activity; Phosphorylated tyrosines serve as docking sites for other signaling molecules.

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 15-54

Binding of SH2-containing proteins to activated PDGF receptors Figure 15-55a

SH2 DOMAINS In ribbon: LH: Isoleu-binding, RHS: P-Y SH2 domains ~100 aa (115 in humans; ~300 SH3 domains) SH2 DOMAINS Figure 15-55b Molecular Biology of the Cell (© Garland Science 2008)

The Ras protein signaling pathway

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.

The regulation of Ras activity GAP = GTPase-activating protein GEF = Guanine nucleotide exchange factor Figure 15-19

Activation of Ras by an activated RTK in the Drosophila eye Figure 15-58 Molecular Biology of the Cell (© Garland Science 2008)

The key step is the recruitment of the Ras-GEF to the plasma membrane. SH2 SH3 The same general principles are true for the Ras pathway in other tissues, and in other organisms (inc. humans). The key step is the recruitment of the Ras-GEF to the plasma membrane.

Ras signaling activates a MAP kinase pathway Figure 15-60

Organization of MAP kinase modules by scaffold proteins Figure 15-61 Molecular Biology of the Cell (© Garland Science 2008)

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)P3, 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)P3. 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.

Generation of PI-based docking sites by PI 3-kinase Figure 15-63 Figure 13-10

Interconversion possibilities that exist between the different forms of PI Figure 13-10

Generation of PI-based docking sites by PI 3-kinase PI(4,5)P2 PI(3)P Phosphoinositide head groups are recognized by protein domains that discriminate between the differently-phosphorylated forms. Figure 15-63 Figure 13-10

Figure 15-21

Pleckstrin homology (PH) domains can mediate binding to PI(3,4,5)P3 PH domain protein cytosol PI(3,4,5)P3 docking site plasma membrane PH domain from PLC-epsilon interacting with IP(3,4,5)P3

PI3K-Akt signaling pathway: Promoting cell survival (mTORC2) Figure 15-64 Molecular Biology of the Cell (© Garland Science 2008)

1. Production of PI(3,4,5)P3 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)P3.

Note that Akt requires two phosphorylation events for activation. 2. Recruitment & activation of Akt mTORC2 Note that Akt requires two phosphorylation events for activation. PDK1 = Phosphoinositol-Dependent Kinase 1

3. Phosphorylation of Bad & inhibition of apoptosis

PI3K-Akt signaling pathway: Promoting cell survival (mTORC2) Figure 15-64 Molecular Biology of the Cell (© Garland Science 2008)

Activation of mTORC1 by the PI3K-Akt pathway Figure 15-65

Potential signaling complexity Figure 15-66 Molecular Biology of the Cell (© Garland Science 2008)

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.

Target cell desensitization Figure 15-29 Molecular Biology of the Cell (© Garland Science 2008)

G-protein-linked receptor desensitization depends on receptor phosphorylation Arrestin binding prevents interaction with G protein. Arrestin may also target receptor to clathrin-coated pits. Figure 15-51 Molecular Biology of the Cell (© Garland Science 2008)