Signal Tranduction

Signal Tranduction

1. Apoptosis

2. GPCR

   • 7 membrane spanning
     • Type IV
       • N out, C in cytosol
   • coupled trimeric G proteins
     • receptor associated with another protein
     • alpha, beta, gamma
       • beta-gamme function as one
       • Alpha inactive when bound to GDP
       • Alpha & gamma linked to cytoplasmic membrane, and held in check by gamma-beta complex
       • 16 alpha 6 beta 12 gamma
     • linked to membrane via lipid-motifs
       • Lipid-anchored proteins at the cytoplasmic face of the PM
   • membrane bound effector protein
   • second messenger in GPCR pathway
   • vision, smell, glycogen breakdown, fuel source, development, muscle relaxation
   • activation
     • hormone binds to 7 transmembranes
       • association of the complex
         • Trimeric complex via lip/tongue- binds to GPCR
           • Conformational change in alpha subunit, promotes dissociation of GDP
             • GEF helps
             • G alpha bound to GTP & linked via lipid-anchor to cytoplasmic membrane
               • Skitters along membrane to find effector protein
                 • GTP bound alpha subunit bind & activates effector protein
                   • Hydrolysis, which promotes alpha subunit to dissociate from effector
                     • Reset mechanism
                       • measure activity in live cells
                         • FRET
                           • Protein-protein interactions in live cells
                           • created artificial experimental condition
                             • taken cDNA, which expresses combination of alpha with CFP & another plasmid, which expresses G-beta fused with YFP
                               • excite with right wavelength
                                 • if these two associate, we excite with optimal excitation light for CFP
                                   • omits an energy light at optimal frequency/wave for YFP- we get yellow fluorescence
                                   • see yellow at PM
                                     • happens when there is no ligand/hormone & the complex is together
                                     • add ligand, you see drop in fluorescence
   • ADENYLYL CYCLASE
     • catalyzes production of cAMP
       • secondary messenger
       • Alpha-S, binds to active adenylyl
         • increased production
   • acetylcholine
     • binds to MAR
       • illicit effect
         • association of G-I-subunit, which binds to activated receptor
           • dissociation of GDP, bind GTP
             • beta-gamma binds to K+ channel
               • Efflux of K+ outside (slows rate of heart muscle contraction
   • Cholera Toxin

3. RTKs

4. Primary messenger

   • first stimuli, quite frequently the initiating signal could be a small molecule
   • Ligand
     • Organic molecules
       • hormones, neurotransmiters
     • Proteins
       • Cytokines, growth factors
     • Ligand-receptor interactions
       • ligand & structure have to match
         • Molecular complementarity
         • often you have conformational change, which lets another receptor snuggle in there as well
           • Dimerization
       • Charge interaction has to be compatible
         • ligand = negative, then receptor = positive
           • Conformational change
             • Dimerization
       • Specificity in affinity
         • Love between the two
         • Equilibrium equation (dissociation constant)
           • K = [R][L]/[R-L]
             • K = affinity, ligand concentration required to bind 50% of the cell surface receptors
               • Ligand really likes receptor (high K), you're not need little cells AND if it has proper shape & charge interactions, it binds effectively
       • monogamous relationship
         • signal specific
       • how does this work?
         • need ligand in pure state
           • Isolate growth factor
             • add bunch of cells in culture
               • some grow like crazy, others don't respond
               • Biochemical stuff but there is an issue
                 • cell surface receptors have hydrophilic part that can bind ligand, but they also have hydrophobic transmembrane protein
                   • Hydrophobic part causes problems for test tube experiments
                 • Use cell line that doesn't respond to growth factors
                   • says that it doesn't express the particular receptor that's going to respond to that ligand
                   • Isolate cDNA from cell that does respond- clone into DNA plasmid vector
                     • cDNA library
                       • introduce it into cell, transcripts enter DNA of non-responding cell
                         • cell proliferates
                           • Take cells from that culture and extract gDNA
                             • PCR- used primers to flank cDNA
                               • Sequence product

5. Receptors

   • Cell surface
     • hydrophilic ligand
       • exist in aqueous environment cannot cross PM transmit signals to inside
       • binds to EC domain
         • conformational change to cytosolic portion
           • activity associated with cytosplasmic domain
             • promote interaction with other types of proteins
               • Effector proteins
                 • proteins that do business or do something that effects overall cell structure, metabolism, function
                 • Transcription factor
                   • gene expression
                 • Cytoskeletal protein
                   • alteration of cytoskeletal protein, which affects cell shape & movement
                 • Metabolic enzyme
                   • activity could be increased/decreased
               • Signaling events
                 • Short term
                   • pre-existing molecules/proteins will be activated following signal tranduction
                 • Longterm
                   • sequential steps, a lot of stuff happens
                     • delay in this related to its relying on modifications or alterations in gene expression
           • interaction with other cytoplasmic proteins
             • same as other one
     • transmembrane protein
     • Cytosolic portion
   • Intracellular
     • Hydrophobic & hormones
       • lipid-soluble, small don't exist in aqueous environ can cross PM & bind inside
       • Carrier proteins help stabilize
         • hydrophobic things don't float around in aqueous environment
     • Cytoplasmic
       • Estrogen, Progesterone, Glucocortoid receptors
     • Nuclear superfam
       • Ultimately go into nucleus & have their activity there
       • DNA binding domain
         • when activated, they bind promoter & regulatory regions of genes & affect gene expression
         • Intracellular receptors also have ligand binding domain
       • EXAMPLE:Steroid
         • Cortisol, retinoic acid, thyroxin
       • Complex ring structure & hydrophobic chains
         • Can cross membranes
       • Glucocorticoid
         • anti-inflammatory
         • Hydrophobic properties
           • Cross the PM
           • Bind to Glucocorticoid receptor
             • found on cytosol because it has inhibitory protein (HSP90
             • HSP90 binds to LBD when there is no ligand
               • receptor doesn't do much
               • When yes glucocorticoid- it binds to LBD
                 • Release of HSP90
                   • Glucocorticoid receptor transported into nucleus
                     • Interacts with another receptor - dimer
                       • Bind to response elements such as DBD- causing it to activate
                         • Bind to glucocorticoid response hormones
                           • sequence of DNA found within the promoter & gene regulatory regions
                           • Cause activation of AD
                             • AD is a little piece of protein that sticks out & helps bring transcriptional machinery, which causes increased/decreased expression
                             • Increase (in most cases) expression
     • 4 forms of signaling
       • Between Cells
         • relies on an area where we produce signal & target for that signal
         • Endocrine
           • Gland produces hormone
             • enter bloodstream
               • Bind to carrier protein
                 • released at target site
                   • exit bloodstream & bind to target receptor
       • Paracrine
         • one cell produces ligand & is in close proximity to another
           • not relying on bloodstream
             • Example: neurons
       • Autocrine
         • own stimulus or ligand
           • has a receptor that ligand
           • cell makes signaling molecules
             • usually transmembrane proteins or is attached to membrane
       • Signalling by PM-attached proteins
         • attached to adjacent target cell
       • Multiple acting at once
         • both endocrine & paracrine (Epinephrine)
         • epidermal growth factor undergoes multiple ones

6. Seconday messengers

   • organic molecules
     • IP3, cAMP, cGMP, DAG, NO, Ca

7. Anmal's dependence on multiple extracellular signal molcules

   • Survival/trophic factors
     • elicit survival signal transduction mechanism Cells need signal to say everything is gucci
     • Other stimuli, telling the cell to divide- mitogenic
     • Signals lost - factor withdrawal or trophic factor withdrawal
       • DEATH
     • pro-apoptotic signals
       • bind to receptor to tell cell to kill itself
         • Cancer = good thing to have
         • Brain cell = bad thing to have

8. Phosphorylation

   • Kinases
     • tryosine, serine, threonine
     • only stick to tyrosine AA
     • dual specficity kinase
       • Serine, Threonine or tyrosine
       • threo/ser
   • alters target activity
   • phosphatases

9. G proteins

   • Bind GTP (when turned on mainly)
     • signaling event
       • activation of effector protein
         • deactivate
           • G protein itself has hydrolytic activity
           • GTPase binds to G protein, increases hydrolytic activity
   • GEF rips out GDP
     • Charge interactions & conformational change
       • Arms close, change in G protein activity