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Unformatted text preview: General principles in signaling between and within cellsSignals: Proteins Peptides Amino acids Nucleotides Steroids Retinoids Fatty acid derivatives gases Signaling proteins Kinases Phosphatases GTP binding proteins Reading for next class: Alberts et al Chapter 16, pgs 965-1010 Extracellular signals bind to plasma membrane receptors or to intracellular receptorsExample of a unicellular organism (yeast) responding to an extracellular signal (a mating pheromone = alpha factor) Analysis of mutants reveals intracellular signaling molecules - pheromone alpha factor + pheromone Different types of inter-cellular signaling- Membrane -tethered signal; adjacent Secreted; short range Electric transmission; long range Secreted; long range Signaling between neighborsCommunity effect/ coordination between cells Cancer cells promote their growth in this way (e.g. secrete and bind growth factors) (autocrine pathway) Quorum sensing in bacteria • Gap junctions allow direct signaling • Only small molecules can pass through.(e.g. ions) • Macromolecules cannot pass through it Extra-cellular signals can elicit a rapid or a slow response in the target cell- e.g. Phosphorylation Ion flows e.g. gene regulation Cells use multiple extra-cellular signals to decide what to doDifferent cells can respond differently to the same signal (e.g. acetocholine). This is because receptors are different, or because the same receptor is coupled to different internal signaling machinery Integrator proteins within cells ultimately determine the cellular response to a combination of signals Different types of signaling molecules (including gases such as nitric oxide)Proteins Peptides Amino acids Nucleotides Steroids Fatty acids gases
Nitric oxide (NO) synthase is the target of pharmaceuticals; it causes blood vessel dilation via muscle cell relaxation: • Example: Heart pills. Nitroglycerine is converted to nitric oxide • Viagra inhibits the cyclic GMP -phosphodiesterase NO has very short half life: 5-10 sec Consequences of NO as pollutant in the environment… Nobel prize 1998 awarded to Furchgott, Ignarro and Murad for discovering NO as a signaling molecule Hormones (steroids) as signaling molecules• Not very soluble often carried by proteins • Membrane diffusible • Far-reaching/systemic The nuclear receptor superfamily binds hormones hormones Binding of hormone causes conformational change in the receptor, exposing active site (e.g. NLS, DNA binding domain, etc.) A hormone signaling cascade- Hormone receptors are often nucleo-cytoplasmic shuttling proteins that function as hormone dependent transcription factors. They are kept inactive by inhibitory proteins that mask their active sites Cascade can be mapped by screening genomic microarrays (by Joe DeRisi, a UCSC undergrad) Three main types of plasma membrane receptors receive the water soluble signals- Fast, typical of neurotransmission act indirectly via trimeric G proteins The ‘signal transducers’ Multipass seven TM domains or channel Most are single pass proteins with extracellular ligand binding domains and cytoplasmic kinase domains A generic map of a signal transduction cascade that utilizes second messenger moleculessecond messenger molecules cAMP calcium soluble cGMP IP3 membrane DAG
Phosphorylation What is the ‘first’ messenger? enzyme Regulation of nuclear import Molecular switches and integrator proteins in intracellular signalingA) Here the molecular switch is the gain or loss of phosphate A) Often part of phosphorylation cascades mediated by serine/threonine kinases or tyrosine kinases B) Involves membrane tethered G -proteins, or small GTP binding proteins; the GTPase activity is used as a clock GEF GAP Molecular switches Integrator proteins coordinate distinct signaling cascades Scaffold proteins ‘focus’ signals to obtain a specific response using generic factors- Avoids cross talk between signaling pathways Increases speed, efficiency, and accuracy Scaffold protein Scaffold protein Often activated by phosphorylation of cytoplasmic domain of receptor Modular assembly of scaffold and adapter proteins- ‘Lego land’’
insulin Evidence of combinatorial biology during evolution…. PTB, SH2 SH3 PH Cells have mechanisms to respond abruptly to gradually-increasing concentrations of a signal- The sharpness of the response increases with increasing number of effectors that must oligomerize to activate the target molecule. This can also be a way for cells to count molecules… Cells use positive feedback and negative feedback loops to stimulate or inhibit signaling- Enzyme remains active after signal is gone Inhibitor dampens or oscillates the signal Five ways to become adapted or desensitized to a signal- Signaling at the plasma membrane through G-protein coupled receptors (GPCR)Ligand dependent activation of a G-protein GPCRs are seven transmembrane domain proteins G-protein: the alpha subunit is a GTPase Ligand dependent conformational change activates G-protein Largest family of signaling molecules (e.g. used for vision, taste, smell); include most knows drug targets. In mice, there are more than one thousand G-coupled receptor devoted to smell! Activation of adenylyl cyclase by the alpha -subunit of a Gs protein- Regulated production Signal ON
Continuous deactivation Signal OFF
A cAMP biosensor… Phosphatase turns signal off Cholera toxin locks alpha in GTP bound form… Activation of phospholipase C by the alpha-subunit of a G protein- IP3 is turned off by dephosphorylation Calcium is pumped out of cells, or back into ER Calcium is also a ‘second messenger’ It affects the activity of a multitude of calcium binding proteins (e.g. calmodulin and CaM kinase) Some G proteins activate ion channels directly (e.g. smell and taste) Signaling through enzyme linked cell surface receptorsSix classes of receptors: • Tyrosine kinases • Tyrosine kinase associated (recruits them) • Tyrosine phosphatases • Serine/threonine kinases • Guanylyl cyclases • Histidine kinase dimer Tyrosine kinases Mode of activation: Dimerization, which lead to auto-phosphorylation Inactive as monomers… activate upon ligand binding and dimerization How signaling proteins cross-link (and thus activate) enzyme-linked cell surface receptors- The ligand is a dimer What happens after activation? The ligand is dimerized by an external scaffold The ligand is localized in apposing cell Activated receptor attracts phosphotyrosine binding proteins that contain SH2 or PTB domains These proteins then go on to propagate the signal. Other phosphotyrosine binding proteins target the receptor for mono-ubiquitination, internalization and degradation Signaling via the Ras GTPase- • Used often for signaling to the nucleus • 30% of human tumors have hyperactive Ras • Tyrosine receptors activate Ras via binding its GEF (Sos) through an adapter protein (Drk /Grb2) GTPase clock
RAF MEK Inactivated by phosphatases ERK Different signaling pathways use the same signaling moleculesHow is cross talk prevented? By using scaffold proteins Parallel pathways for intracellular signaling activated by G protein linked receptors and tyrosine kinases- ...
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