1. G-protein-linked receptors Brian Pierchala BIO402/502 MBOC4 Chapter 15

2. G-protein-linked receptors **These are also calld G-protein-coupled receptors (GPCRs). **GPCRs comprise the largest family of cell surface receptors. In mice, there are 1000 different receptors involved in smell alone. **A single ligand often binds to more than one GPCR. The neurotransmitter serotonin, for example, has at least 15 GPCRs. **Half of the known medications we take work through GPCRs. **All GPCRs have the characteristic 7 transmembrane domains with the same topology: the N- terminus is extracellular and typically comprises most of the ligand binding domain. The C- terminus is intracellular. **All GPCRs signal via the use of G-proteins.

3. Inactive heterotrimeric G-protein **Heterotrimeric G-proteins are tethered to the membrane via lipid anchors. Both the alpha and gamma subunits have covalent lipid modifications. **In an inactive state, the alpha subunit binds GDP and is a GTPase. The beta subunit, which binds to the alpha subunit, locks the alpha subunit in an inactive conformation. **There are several types of G-proteins, each of which bind to a particular set of GPCRs, and to a particular set of intracellular signaling molecules.

4. G-protein activation 1.When not activated, the alpha subunit binds GDP. 2.When the ligand binds to the GPCR it changes conformation and, in turn, alters the conformation of the G protein. 3.Altering the conformation of the alpha subunit allows it to exchange GDP for GTP. 4.The binding of GTP to the alpha subunit causes it to dissociate from the beta subunit. 5.These are now two independent entities: the alpha subunit and the beta-gamma (  ) subunit. 6.GTP binding and dissociation of the  subunit causes the alpha subunit to adopt a new conformation. The alpha subunit can now bind to intracellular signaling proteins. 7.The  subunit does not change conformation, but its dissociation from the alpha exposes this face of the beta subunit. It now can also interact with signaling molecules.

5. Inactivation of G proteins 1.The active alpha subunit binds to its target protein and activates it. Common targets of alpha subunits are enzymes and ion channels. 2.The internal GTPase activity of the alpha subunit cleaves the GTP to GDP. 3.This conversion to GDP inactivates the alpha subunit, causing it to dissociate from its target protein. 4.The inactive alpha subunit can then associate again with the  subunit, reforming the inactive G protein. **G proteins are usually active for very short periods of time (seconds). **Alpha subunits have intrinsically weak GTPase activity, and on their own would take minutes to hydrolyze their GTP. **GTPase activating proteins (GAPs), when bound to the alpha subunit, enhance the GTPase activity tremendously. GAPs are critical negative regulators of G proteins. **Regulator of G protein signaling (RGS) proteins are a large family of GAPs that are thought to be required for turning off G protein cascades in ALL eukaryotes. **Each RGS protein has preferred alphas that they regulate.

6. Some G proteins signal via cyclic AMP (cAMP) **Stimulatory G proteins (G s ) activate an enzyme called adenylyl cyclase, which produces cAMP from ATP. Adenylyl cyclase is a transmembrane protein with its enzymatic region in the cytoplasm. **This image depicts a neuron that has been stimulated with serotonin, which is activating a GPCR that is coupled to G s. The image shows the monitoring of cAMP levels with a fluorescent method. Red indicates an increase of cAMP by 20-fold within 20 seconds. **There are inhibitory G proteins (Gi) that inhibit adenylyl cyclase, thereby stopping the production of cAMP. **cAMP phosphodiesterases hydrolyze cAMP to AMP, thereby counter-acting the production of cAMP. Often cAMP levels rapidly decline after production due to cAMP phosphodiesterases.

7. Production of cAMP 1.Adenylyl cyclase produces cAMP by removing two phosphate groups from ATP. 2.The phosphates are removed as pyrophosphate (P-P). 3.Along with the removal of pyrophosphate the molecule is cyclized. 4.cAMP phosphodiesterase then hydrolyzes cAMP to AMP. Cholera toxin: inactivates the GTPase activity of the G s alpha subunit, thereby keeping it active. This causes oversecretion of chloride ions and water into the gut (severe diahrrhea). Pertussis toxin: this toxin inactivates the alpha subunit of G i. This blocks its ability to negatively regulate its targets (whooping cough).

8. Most effects of cAMP are mediated by protein kinase A (PKA) 1.PKA is activated by cAMP and mediates the majority of cAMP effects. 2.There are many different substrates of PKA in different cells, which may explain why rises in cAMP in different cell types result in very different responses. 3.The inactive form of PKA is a heterotetramer of two catalytic (kinase) subunits and two regulatory subunits. 4.The binding of cAMP to the regulatory subunits causes a conformational change that releases the catalytic subunits. 5.The release of the catalytic subunits activates them, allowing them to phosphorylate their substrates on serine and threonine residues.

9. Rapid and Slow responses to PKA activation **Some of the effects of PKA activation are rapid. Example: the stimulation of glycogen breakdown to glucose in muscle cells. This occurs by the direct phosphorylation of proteins involved in glycogen metabolism. This provides glucose for energy production in muscle cells within seconds. **Some of the effects of PKA are slower. Example: the activation of gene expression, such as the somatostatin hormone. Activated PKA can translocate into the nucleus. There it phosphorylates the transcription factor CREB (cAMP response element binding protein). 3.When CREB is phosphorylated it binds to the cAMP response element (CRE). 4.CREB-binding protein (CBP) binds to phosphorylated CREB and activates transcription of genes that contain CRE sequences, such as the somatostatin gene. 5.Many cAMP-induced genes contain CRE sequences and are regulated by CREB and CBP.

10. Production of inositol phospholipids **Phosphatylinositol (PI) 4-phosphate and PI 4,5 bisphosphate are produced by the sequential actions of PI kinase and PIP kinase, respectively. **PIs exist in the inner leaflet of the plasma membrane. **PI 4,5 bisphosphate is especially important because its breakdown produces two different second messengers. **PI 4,5 bisphosphate is the least abundant of the PIs, and accounts for only 1% of total phospholipids.

11. Phospholipase C-  is critical for GPCR signaling 1.The G protein q (G q ) alpha subunit activates the enzyme phospholipase C-  (PLC-  ) in a manner similar to how G s activates adenylyl cyclase. 2.Activated PLC-  cleaves PI 4,5 bisphosphate to produce diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ). 3.Importantly, both DAG and IP 3 are second messengers that activate distinct intracellular signaling molecules. 4.IP 3 is a small, water-soluble molecule that readily diffuses through the cytosol. 5.DAG remains embedded in the plasma membrane, but like other PM lipids, can diffuse laterally through the membrane.

12. The targets of IP 3 and DAG IP 3 : When IP 3 diffuses to the membrane of the ER it binds to IP 3 -gated calcium release channels (IP 3 Rs) in the membrane, triggering their opening. **IP 3 Rs release calcium from the ER into the cytosol, rapidly increasing the concentration of calcium in the cytosol. Ca 2+ is perhaps the most common second messenger in cells. **Calcium levels are quickly reduced by channels that pump it out of the cell, and by the inactivation of IP 3 by dephosphorylation and other means. DAG: DAG has two signaling functions. First, it can be further cleaved to arachidonic acid, which can initiate a complex cascade of lipid messengers. Second, DAG can activate a serine/threonine kinase called protein kinase C (PKC). **PKC requires both Ca 2+ and DAG, along with membrane phospholipids, to be activated. **PKC has numerous protein substrates that are unique from PKA.

13. Calcium is a ubiquitous second messenger 1.Ca 2+ is a second messenger derived from the activation of many types of cell surface proteins, not just GPCRs. 2.Calcium levels in the cytosol is very low (10 -7 M) and very high extracellularly (10 -3 M). Calcium levels are also high in the ER. 3.Therefore, modest amounts of calcium entering the cytosol can change the concentration very rapidly by 10-20 fold. 4.This is an image of an egg cell that is being fertilized by a sperm. This process triggers a wave of calcium release from the ER from the site of contact with the sperm. This wave of calcium triggers the beginning of embryonic development.

14. Calmodulin 1.Calmodulin is a Ca 2+ binding protein that has 4 high affinity binding sites for Ca 2+. 2.Calmodulin is extremely abundant in cells and accounts for as much as 1% of total protein. 3.Binding of calcium causes a conformational change in calmodulin. 4.At least two or more Ca 2+ must bind before calmodulin changes conformations, making it behave like a switch to increasing concentrations of calcium. 5.Calmodulin has no enzymatic function, and instead binds to target proteins and alters their confirmation (as well as its own). 6.One of the most important group of calmodulin targets is the Ca 2+ /calmodulin-dependent protein kinases (CaM-kinases).

15. CaM-kinase II 1.CaM-kinase II is composed of a large complex of about 12 subunits of CaM- kinase II. For simplicity, only one is shown here. 2.Upon Ca 2+ /calmodulin binding, CaMKII changes conformation and is activated. 3.Upon activation, CaMKII autophosphorylates itself on an autoinhibitory domain. This phosphorylation event sustains CaMKII activity without Ca 2+ /calmodulin being present in two ways. First, it locks Ca 2+ /calmodulin binding to it such that it will not dissociate without the prolonged return to normal calcium levels. Second, it converts the enzyme to a calcium independent form. 4.After this occurs, CaMKII can only be inactivated if all of the subunits are dephosphorylated by phosphatases (overriding the CaMKII kinase activity).

16. CaMKII is a “frequency decoder” for calcium waves 1.During low-frequency calcium spikes (or oscillations), CaMKII does not remain active long enough before the next calcium spike, and there is no overall difference in subsequent CaMKII activations. 2.During high frequency calcium waves, the amount of CaMKII activity ratchets up upon each successive calcium spike because it never fully inactivated. This “ratcheting” of CaMKII activity will continue until all 12 subunits are fully activated. It will remain active for a long period of time, even after the calcium spikes stop. **This is a type of “cellular memory” because CaMKII can “remember” prior calcium spikes. **CaMKII is critical for spatial memory in the brain (where CaMKII is as much as 2% of brain protein!!). It is this quality of calcium frequency decoding that enables it to encode memories.

17. GPCR desensitization **Cells desensitize, or adapt, when exposed to high levels of ligand for a long period of time. There are 3 mechanisms of desensitization at the level of the GPCR: 1.Receptor inactivation: The GPCR becomes modified such that it can no longer interact with its G protein. 2.Receptor sequestration: The GPCR can be internalized and transported to an interior compartment of the cell such that it no longer is exposed to ligand. 3.Receptor downregulation: The receptor can be degraded by lysosomes after it is internalized. G-protein-linked receptor kinases (GRKs): phosphorylate GPCRs upon their activation on multiple serines and threonines. This phosphorylation leads to binding of arrestin to the active GPCR. Arrestin triggers desensitization by 1). inhibiting the binding of the G protein to the GPCR and 2). by acting as an adaptor protein for the internalization of the receptor. Whether the receptor is sequestered or degraded depends upon may factors.