1. G-protein-Couped receptor Liu Ningsheng 12/3/2010

2. Cell structure

3. (A) Most signal molecules are hydrophilic, they bind to cell-surface receptors, which in turn generate signals inside the target cell. (B) Some small,signal molecules, by contrast, diffuse across the plasma membrane and bind to receptor proteins inside the target cell— either in the cytosol or in the nucleus The binding of extracellular signal molecules to either cell-surface or intracellular receptors.

4. The signal molecule usually binds to a receptor protein that is embedded in the plasma membrane of the target cell and activates one or more intracellular signaling pathways mediated by a series of signaling proteins. Finally, one or more of the intracellular signaling proteins alters the activity of effector proteins and thereby the behavior of the cell. A simple intracellular signaling pathway activated by an extracellular signal molecule.

5. In this example, a series of signaling proteins and small intracellular mediators relay the extracellular signal into the nucleus, causing a change in gene expression. A hypothetical intracellular signaling pathway from a cell-surface receptor to the nucleus.

6. Three classes of cellsurface receptors.

7. Overview of seven major classes of cellsurface receptors

8. Although one type is activated by phosphorylation and the other by GTP binding, in both cases the addition of a phosphate group switches the activation state of the protein and the removal of the phosphate switches it back again. Two types of intracellular signaling proteins that act as molecular switches.

9. GPCRs that bind protein ligands have a large extracellular domain formed by the part of the polypeptide chain shown in light green. This domain, together with some of the transmembrane segments, binds the protein ligand. Receptors for small ligands such as adrenaline have small extracellular domains, and the ligand usually binds deep within the plane of the membrane to a site that is formed by amino acids from several transmembrane segments. A G-protein-coupled receptor (GPCR).

10. All receptors of this type have the same orientation in the membrane and contain seven transmembrane -helical regions (H1–H7), four extracellular segments (E1–E4), and four cytosolic segments (C1–C4). The carboxyl-terminal segment (C4), the C3 loop, and, in some receptors, also the C2 loop are involved in interactions with a coupled trimeric G protein. Schematic diagram of the general structure of G protein–coupled receptors.

11. (A) Note that both the a and the g subunits have covalently attached lipid molecules (red) that help bind them to the plasma membrane, and the a subunit has GDP bound. (B) The three-dimensional structure of an inactive G protein. the G protein that operates in visual transduction. The a subunit contains the GTPase domain and binds to one side of the b subunit, which locks the GTPase domain in an inactive conformation that binds GDP. The g subunit binds to the opposite side of the b subunit, and the b and g subunits together form a single functional unit. (B) The structure of an inactive G protein.

12. Binding of an extracellular signal to a GPCR changes the conformation of the receptor, which in turn alters the conformation of the G protein. The alteration of the a subunit of the G protein allows it to exchange its GDP for GTP, activating both the a subunit and the bg complex, both of which can regulate the activity of target proteins in the plasma membrane. The receptor stays active while the external signal molecule is bound to it, and it can therefore catalyze the activation of many molecules of G protein, which dissociate from the receptor once activated (not shown). In some cases, the a subunit and the bg complex dissociate from each other when the G protein is activated. Activation of a G protein by an activated GPCR.

13. The G and G subunits of trimeric G proteins are tethered to the membrane by covalently attached lipid molecules. Following ligand binding, dissociation of the G protein, and exchange of GDP with GTP (steps 1 – 3 ), the free G·GTP binds to and activates an effector protein (step 4 ). Hydrolysis of GTP terminates signaling and leads to reassembly of the trimeric form, returning the system to the resting state (step 5 ). Binding of another ligand molecule causes repetition of the cycle. In some pathways, the effector protein is activated by the free G subunit. model for ligand-induced activation of G protein– coupled receptors.

14. Moives http://www.celanphy.science.ru.nl/Bruce%20web/Flash%20Movies.htm

15. Four Major Families of Trimeric G Proteins*

17. (a)Schematic diagram of mammalian adenylyl cyclases. The membrane-bound enzyme contains two similar catalytic domains on the cytosolic face of the membrane and two integral membrane domains, each of which is thought to contain six transmembrane helices. (b) Three-dimensional structure of Gs·GTP complexed with two fragments encompassing the catalytic domain of adenylyl cyclase determined by x-ray crystallography. Structure of mammalian adenylyl cyclases and their interaction with Gs·GTP.

18. The binding of cyclic AMP to the regulatory subunits of the PKA tetramer induces a conformational change, causing these subunits to dissociate from the catalytic subunits, thereby activating the kinase activity of the catalytic subunits. The activation of cyclic-AMP-dependent protein kinase (PKA).

19. Effect on adenylyl cyclase

20. Ligand binding to Gs-coupled receptors causes activation of adenylyl cyclase, whereas ligand binding to Gi-coupled receptors causes inhibition of the enzyme. The G subunit in both stimulatory and inhibitory G proteins is identical; the G subunits and their corresponding receptors differ. Ligand-stimulated formation of active G·GTP complexes occurs by the same mechanism in both Gs and Gi proteins. Gs·GTP and Gi·GTP interact differently with adenylyl cyclase, so that one stimulates and the other inhibits its catalytic activity. Hormone-induced activation and inhibition of adenylyl cyclase in adipose cells.

21. The binding of an extracellular signal molecule to its GPCR activates adenylyl cyclase via Gs and thereby increases cyclic AMP concentration in the cytosol.. How a rise in intracellular cyclic AMP concentration can Alter gene transcription.

22. Receptor stimulation ( 1 ) leads to activation of PKA ( 2 ). Catalytic subunits of PKA translocate to the nucleus ( 3 ) and there phosphorylate and activate the transcription factor CREB ( 4 ). Phosphorylated CREB associates with the co-activator CBP/P300 ( 5 ) to stimulate various target genes controlled by the CRE regulatory element. See the text for details. Activation of gene expression following ligand binding to Gs protein–coupled receptors.

23. The more steps in such a cascade, the greater the signal amplification possible. Amplification of an external signal downstream from a cell-surface receptor.

24. How GPCRs increase cytosolic Ca2+ and activate PKC.

25. IP3/DAG pathway & the elevation of cytosolic Ca2.

26. In resting cells, Tubby is bound tightly to PIP2 in the plasma membrane. Receptor stimulation leads to activation of phospholipase C, hydrolysis of PIP2, and release of Tubby into the cytosol ( 1 ). Directed by two functional nuclear localization sequences (NLS) in its N-terminal domain, Tubby translocates into the nucleus ( 2 ) and activates transcription of target genes ( 3 ). Activation of the Tubby transcription factor following ligand binding to receptors coupled to Go or Gq.

27. A GRK phosphorylates only activated receptors because it is the activated GPCR that activates the GRK. The binding of an arrestin to the phosphorylated receptor prevents the receptor from binding to its G protein and also directs its endocytosis (not shown). Mice that are deficient in one form of arrestin fail to desensitize in response to morphine, for example, attesting to the importance of arrestins for desensitization. The roles of GPCR kinases (GRKs) and arrestins in GPCR desensitization.

28. Further Reading 1.Molecular Biology Of The Cell. Alberts. (Chapter 15) 2.Molecular Cell Biology. Lodish (Chapter 13)

29. Extracellular signaling molecules regulate interactions between unicellular organisms and are critical regulators of physiology and development in multicellular organisms. Binding of extracellular signaling molecules to cell-surface receptors triggers intracellular signal-transduction pathways that ultimately modulate cellular metabolism, function, or gene expression Receptors bind ligands with considerable specificity,which is determined by noncovalent interactions between a ligand and specific amino acids in the receptor protein The level of second messengers, such as Ca2, cAMP,and IP3, increases or occasionally decreases in response to binding of ligand to cell-surface receptors. These nonprotein intracellular signaling molecules, in turn, regulate the activities of enzymes and nonenzymatic proteins. Trimeric G proteins transduce signals from coupled cellsurface receptors to associated effector proteins, which are either enzymes that form second messengers or cation channel proteins. Take-home Message

30. Signals most commonly are transduced by G, a GTPase switch protein that alternates between an active (“on”) state with bound GTP and inactive (“off”) state with GDP. The and subunits, which remain bound together, occasionally transduce signals. Gs, which is activated by multiple types of GPCRs, binds to and activates adenylyl cyclase, enhancing the synthesis of 3,5-cyclic AMP (cAMP). cAMP- dependent activation of protein kinase A (PKA) mediates the diverse effects of cAMP in different cells. The substrates for PKA and thus the cellular response to hormone-induced activation of PKA vary among cell types. Simulation of some GPCRs and other cell-surface receptors leads to activation of phospholipase C, which generates two second messengers: diffusible IP3 and membrane-bound DAG IP3 triggers opening of IP3-gated Ca2 channels in the endoplasmic reticulum and elevation of cytosolic free Ca2. In response to elevated cytosolic Ca2, protein kinase C is recruited to the plasma membrane, where it is activated by DAG