CBIO 3400 Week 3 Study Questions 1 Compare and contrast how GPCR and RTK utilize nucleotide triphosphates as the Activated RTK cid 1 s Assemble Large energy source to turn on downstream signaling pathways RTKs use nucleotide triphosphates to auto phosphorylate and then trans phosphorylate themselves to activate Once activated RTKs assemble large multi molecular complexes that create a signaling cascade the proteins that make up this large complex relay the signal downstream Binds Multimolecular Complexes Protein Motifs Src Homology 2 SH2 phosphorylated tyrosine Protein Tyrosine Binding PTB Binds phosphorylated tyrosine SRC Homology 3 SH3 Bind proline rich regions PH Domain Bind Pleckstrin Homology GPCRs use nucleotide triphosphates to activate their and complexes both of which can regulate the activity of target proteins in the plasma membrane These activated complexes bind and activate an enzyme such as adenylyl cyclase which causes the production of a second messenger such as cAMP that can bind to and activate a target such as Protein Kinase A phosphorylated inositol lipid SH2 2 Use one signaling pathway as an example to explain how RTK and G protein are associated in a single signaling pathway The signaling pathway in the fly eye uses both RTK and G protein to transmit a signal 1 3 What mechanisms exist to down regulate turn off signaling from GPCR A GPCR can turn itself off with GAP GTPase activating protein which stimulates GTPase by the activated subunit such as and causes phosphodiesterase to cleave cAMP This causes the protein kinase to turn off and effectively turns off GPCR signaling with the reassociation of the and subunits on the G protein A GPCR can also turn itself off by being phosphorylated at multiple sites attracting a protein such as arrestin and desensitizing the GPCR adrenergic receptor kinase BARK 4 Compare and contrast signaling through Rhodopsin versus signaling through the New findings also implicate arrestin in other functions Retina Promote endocytosis Activation of MAP kinase RTK by cross talk Scaffold protein to organize signaling pathways Lefkowitz and Shenoy Science 308 512 517 2005 adrenergic receptor Adrenalin Pathway Adrenergic Receptor The signal adrenalin binds to the adrenergic receptor This causes the and subunits to dissociate at which point the s subunit binds to and turns on the enzyme adenylyl cyclase Adenylyl cyclases activates the second messenger cAMP freeing the catalytic units cAMP then binds to Protein Kinase A G Protein Signaling in the Retina Rhodopsin Light is the signal that activates the receptor rhodopsin This activates the G protein transducin which stimulates a cGMP phosphodiesterase The cGMP phosphodiesterase cleaves cGMP causing a decrease in cGMP This decrease is effectively the second messenger because cGMP is the ligand that activates ligand gated Na channel The decrease in cGMP causes the pathway to reach its target closing of the Na channel 5 IP3 plays an important role in Calcium dependent signaling Explain where and how IP3 is synthesized and how it is related to Calcium dependent signaling IP3 is synthesized in the cytosol when phospholipase C cleaves PIP2 to produce diacylglycerol DAG and inositol triphosphate IP3 IP3 is an important part of Ca2 dependent signaling because IP3 can travel a much longer distance in the cytosol than Ca2 can IP3 travels to the lumen of the endoplasmic reticulum where it binds and opens an IP3 gated Ca2 release channel The Ca2 can then travel a shorter distance to bind and activate Protein Kinase C See related image on next page 2 GPCRs Activate PLC 6 Describe the differences in function protein interactions and regulation of Protein PLC Kinase A Protein Kinase B and Protein Kinase C Protein Kinase A Function phosphorylates specific serines and threonines on selected target proteins including intracellular signaling proteins and effector proteins thereby regulating their activity Protein Interactions consists of a complex of 2 catalytic subunits and 2 regulatory subunits binding of cAMP to regulatory subunits alters their conformation causing them to dissociate from the complex the released catalytic subunits are thereby activated to phosphorylate specific target proteins Regulation in unstimulated cells the phosphodiesterase keeps the local cAMP concentration low so bound PKA is inactive in stimulated cells cAMP concentrations rapidly rise overwhelming the phosphodiesterase and activating the PKA Protein Kinase B also called Akt Function regulates cell survival and metabolism by binding and regulating many downstream effectors promotes cell growth and survival PI 3K Pathway Promotes Growth Survival Protein Interactions PKB activates mTOR to promote cell growth Regulation involved in the PI 3 K PKB mTOR pathway activation requires binding of the PH mTOR is mammalian Target Of Rapamycin protein kinase domain to 3 phosphoinositides and phosphorylation of PKB by PDK1 Protein Kinase B Activates mTOR to Promote Cell Growth Grpwth factors bind RTKs and stimulate protein synthesis Activate PI 3K Activate Akt Inactivate Tsc 2 Tsc2 is a GAP That inactives Rheb Rheb GTP actives mTOR mTOR promotes cell growth by stimulating protein synthesis ribosome synthesis and nutrient uptake Summarize PI Signaling 3 Protein Kinase C Function tumor promoter in presence of mutagen makes mutagen work better Protein Interactions activates Na H antiport 5P tase cleaves phosphate from carbon 5 of IP3 which is capable of opening a Ca2 channel to produce IP2 which cannot open a Ca2 channel Regulation lipids contribute to activation diacylglycerol DAG phosphatidyl serine synthesized in inactivated state some forms activated by Ca2 some forms Ca2 independent multiple sites of phosphorylation including auto phosphorylation PKC anchoring proteins control localization 4