General Pathways PDF

Questions and Problems #11 RTKs and other catalytic receptors Adaptor (adapter) proteins Typically, the RTK signaling pathways start with the binding of ligands (i.e., growth factors) and the induction of receptor dimerization. The dimerization facilitates the trans- phosphorylation of RTKs, and t...

Questions and Problems #11 RTKs and other catalytic receptors Adaptor (adapter) proteins Typically, the RTK signaling pathways start with the binding of ligands (i.e., growth factors) and the induction of receptor dimerization. The dimerization facilitates the trans- phosphorylation of RTKs, and the phosphorylated tyrosine residues recruit and activate downstream signaling molecules. These molecules directly bind the RTKs or are indirectly recruited by adaptor proteins. Adaptor proteins lack enzymatic activity; they link proteins. The adaptor proteins are classified into two groups based on structure and function. Group 1 is the docking proteins with multiple tyrosine phosphorylation sites that bind downstream signaling proteins. Examples: GRB2-associated binding protein (GAB), insulin receptor substrate (IRS), etc. These adaptor proteins often contain membrane localization domains. Group 2 is the adaptor proteins with SH3 and/or SH2 domains only. They do not have membrane localization structures or phosphorylation sites. One example is GRB2 which has one SH2 domain and two SH3 domains. Grb2 binds to RTKs and non-receptor tyrosine kinases (e.g., FAK). GRB2 also binds SOS1, a guanine-nucleotide exchange factor (a GEF) of RAS. Activated RAS (i.e., RAS-GTP) can initiate the downstream mitogen-activated protein kinase (MAPK) cascade. GRB2 can also bind the docking protein IRS1. Molecular Biology Concepts 8. L-Arginine and NO Generation in Vasodilation L-arginine serves as a substrate for endogenous nitric oxide (NO) generation. The endothelial nitric oxide synthase (eNOS) catalyzes the reaction. NO relaxes the smooth muscle cells in arteries, the arteries increase their diameter (vasodilate), and therefore, the blood supply increases. When blood vessels dilate, the blood flow is increased due to decreased vascular resistance. Therefore, dilation of arteries and arterioles decreases arterial blood pressure and heart rate. At the molecular level, NO binds soluble (cytosolic) guanylyl cyclase to stimulate cGMP production. cGMP activates PKG (cGMP-dependent protein kinase). PKG activates myosin phosphatase which allows the release of calcium from intracellular stores in smooth muscle cells (this information is not included in the lectures). This in turn leads to the relaxation of the smooth muscle cells. Phosphodiesterases convert cyclic nucleotide monophosphate to nucleotide monophosphates. 9. GTPase Activity and Ras Signaling The GTPase activity is intrinsic to Ras and other small GTPases, as well as to all other G- proteins (think of G-alpha). GAP proteins accelerate the hydrolysis of GTP to GDP on GTP- bound proteins such as Ras. In the absence of a signal, the lack of activation of GTPase activity may result in background activity. Thus, the balance between GTP-RAS and GDP- RAS would be skewed in the direction of GTP-RAS. In the presence of a growth factor signaling through an RTK (e.g., EGFR), the lack of RAS-GTPase activity would result in sustained RAS signaling, however, there will be a saturation point reached once all RAS molecules are bound to GTP. 10. Effects of RTK Mutations (A) This will be an inactive RTK because the ligands cannot bind it. There will be no effect on the signaling pathway of the cells. (B) This will be an inactive RTK because the kinase domain and the tyrosine phosphorylation sites will be missing. However, endogenous signaling might be suppressed because ligands may be sequestered (buffered) to bind to the predominant mutant at the cell surface. This is a dominant-negative effect. 13. RAS-GEF and RAS-GAP Impact on RAS Activity (A) Activation of a RAS-GEF will activate RAS (positive feedback) and increase the RAS-GTP form levels; therefore, this activity will increase MEK activity downstream. (B) Activation of a RAS-GAP will decrease the activity of RAS (negative feedback) by converting it to RAS-GDP and therefore, will decrease the activation of MEK downstream.