molecular bio test 2

• amino acids
• peptides
• soluble protein
• steroids - can diffuse through the pm and 
•     bind to intracellular receptors

• RNA
• (hormones)

need an integral membrane protein to get through

• extracellular
• membrane spanning
• facing cytosol 
•  - GPCRS (G-protein coupled receptors)

Long distance hormones (secreted by signaling molecules)(endocrine cell)

• Paracrine
•  nerve cell -> nerve cell
• nerve cell -> muscle cell
• (growth factor)

Autocrine -self signaling (tumor)

• low abundance of particular receptor req 
• -> highspec

• 1. Weak multiple noncovalent forces
• 2. Ionic
• 3. Molecular complementary
• 4. van der waals

• kon - rate of formation of complex
• koff rate of dissociation of ligand from receptor
• R+C <=> RL

• @Equilibrium
• koff <=> kon

k_D =rate of dissociation

KD = [R][L]/[RL]

measure of the affinity of receptor for the ligand

• 1/2 receptors are bound w/ ligand
•  ->km = 1/2 vmax

• [I¹²⁵] = the amt of radioactive insulin bound to receptor

• molecules/cell bound vs
• [I125 insulin] - the amount of extracellular insulin added to the medium

For total binding: add saturated amounts of radio labeled insulin

For specific binding saturate w/ 100 fold unlabeled insulin

for non specific binding = add labeled insulin

• IP = labeled high affinity
• EP = low affinity
• top dashed line - kdip = kdep
• vertical the concentration @ which alophrenol
• binding is inhibited by 50%
• add IP, continuously add EP at what concentration is it outcompeting

mimic - induces a normal response/receptor

bind + elicit no response

kD = must be higher than instimulated hormone in blood.

• Suppose normal conc. of insulin in the blood
• is 5x10^-12M

kD = 1.4x10^-10 M

• For binding insulin
• -> [RL]/([RL] +[R])
• = .03444
• 3%

• increases conc. of insulin 5 fold 
• 2.5x10^-11 M -> 15%

• 50% receptors are bound 
• increase conc 5 fold # of receptors bound 
• goes up to 66%

• -Feed back mechanisms
• -energy

Immuno affinity chromatograph

Trimeric G proteins

-GTP hydrolysis

• needs GAP (activate prot)
• and GEF (exchange factor)

kinases + phosphatases

1. Receptor w/ 7 transmembrane spanning domains

• 2. coupled trimeric G protein (alpha,beta, gamma)
• 3. Effector (membrane bound)
• 4. Feed back regulation/ desensitization of the pathway

G -protein coupled receptors are a large and diverse famility of proteins who's primary funtion is to transduce extracullular stimuli into

G-protein complex

• 3-subunits
•   active state replaces GDP w/ GTP

12 or viruses to generate all the g-protein complexes used in these exp

the various isolated G-protein complexes mutated

various isolated G-protein complexes where then tested to show binding interactions 

various combinations of different alpha, beta, and gamma subuntits

why is it that when they added the non-hydrolizable version why is that it decreased txn from 2 ->1 

GTP <- adds more weight so two bands

• Found new-protein complex, stronger
• Different combinations expressed disticted interactions showing that gene interactions are highly specific compared to other.

• 7 trans membrane segments
• -> 4 cytosolic segments
• -> 4 extracellular segments

mostly hydrophobic amino acids stay anchored

1. epinephrine (stress hormone) binds to the epinephrine receptor -> release glucose

2. Trimeric G-protein      

•  alpha/beta/gamma } resting
• Gapha and Ggamma are anchored to membrane  
•  Conformation change to cytosolic segment of receptor allows (binding?) to the trimeric G-protein

• 3. Binding of receptor to trimeric G-protein results in release of GDP   
• No hydrolyzable GTP to test if it works

• 4. Trimeric G protein Galpha subunit conformational change that opens GTP binding site
•       GTP bind - dissociation of Gy from     Gapha

Active GTpGapha is short lived (minutes or less)

GTP -> GDP + pi

GTP hydrolysis is enchanced when Galpha GTP interacts w/ the effector (Phosolipase C Adenylyl cyclases) (the effector acts as GAP)

• Acetylcholine ligand 
• -> pancreatic cells 
• ->  ^Ca++        
•    -> muscle contraction       
•    -> hormone secretion 
•    -> secretion of digestive enzymes

Adenylyl cyclase the next step after trimeric protien

Overexpression of wnt-1 gene has been found in a number of ovarian cancers

caused by mammary mouse tumor virus

wnt is involved in limb pattern, organs

• Beta calanin * membrane cytoskeleton anchor protein transcriptional activator
• (kinase) GSK3- phosphorylates Beta catenin 
•    -> Negative effects

• Ap C - interacts w/ Beta catenin complex
• Axin - interacts w/ Beta catenin complex

(Diagram)

sdaf

Like wnt-hedgehog has membrane multiple spanning regions in its receptors

• Ca++  is 10^-7 it cytosol, increases 100 fold when GPCR is activated, from ER 
• -influx of Ca++ from external environment

*PKC phosphorilates Ca++ channel in extracellular memebrane

increase in Ca++ in muscle cells = contractions

increase in Ca++ -> release of neurotransmitters

pKA phosphorilates lots of targets opens Ion channels

• -Ras
• -Raf
• -Sar1

• Trimeric Gproteins (alpha-GDP, beta, gamma) } receptor GEF, effector
•           molecule-GAP

• Second messengers
• AkAp15 - tethered on the membrane next to a gated Ca++ channel
•      - binds to (pKA)
•      - PDA is adjacent to AkAp15
•           - can turn stuff off camp-(prot)-(prot)

A g-protein receptor that regulates

ok

Ab specifically for kinase and bind to beads (protein A)

Mix beads w/ cytosolic extract or nuclear extract

• centrifuge 
• wash w/ salt solution
• Left w/ kinase/Ab + any proteins that bind to kinase

• incubate
• kinase/Ab/proteins/beads
• w/ Y32pATP
• Histone H1 (substrate) 
• -kinase activity->
• I  PAGE gel
• Autoradiography
• II IP substrate (Histone H1)
• w/ Ab read w/ scintillation counter

• + small Gprotein -> (PAK1)
• + binding protein (PAK1)(PBD)
• antibody (beads) to the Gprotein PBD -> proteins
• binding domain
• + extracts
• recover by centrifuging
• western blot alpha G protein ab

• -are involved in growth processes 
• -cell growth + differentiation +feedback   
•    -EGF, PDGF etc..
• -> MAP kinase cascade   
• ->txn activation           
• *Thinking about chromatin remodeling activation of txn factors repression

• Always in dimer form
• intracellular that is phosphorilated
• -> Exterior portion (ligand binding site)
•                                *autophosphorylation
• -> interior                  *crossphosphorilation
• -> phosporilation sites *complimentary
•                      sites 1 phosphorilation                                 induces phosphorilation of            site 2 ect vio confrontational change 
• attracts
• sh3 domain {->Serine/Threonine or tyrosine
• containing { -> dowstream elements ->
• protein      {     sh2(binding domain)/PTB ->
•               {(protein tyrosine binding domian)

• -> Ligand can already be dimer
• > Signal ligand binds,induces conformational change of single receptor -> receptor then dimerizes, increase affinity of receptor (2) to ligand (2)

• -> receptor can already be a dimer
• Turn off by generation a AB that blocks binding site

• Turn off by mutate kinase domain
•    i
•     Turn off by mutate kinase domain

• universal steps for al RTKS
• -> in resting unstimulated state untrinsic kinase activity is low.
• -> in ligand bound dimeric stage 
•    - the kinase ins one subunit phosphorilates the Y (tyrosine) residue in the other subunit
• (activation)
• -> phorphorilates tyrosine residues result in binding of ATP (insulin) or proteins (EGF/
• FGF (type of receptor) to the downstream phosphorylates Y residues

• SHP1 -> cytokine receptors
• epo receptor - JAK kinase 
• -ineffective when it is not phosphorilated
• -SH2 domains active SHP1 binds to and dephosphorilation JAK 

Receptor resting state

• SHP1 ->  
• SH2 Domain { (prot) <- phosphatase domains

• SOCS Negative regulate ->STATS
• STATS - transcription factor activated
• by cytokine receptors (JAK kinase)
• 1. SH2 domains of SOCS binds to phopho tyrosine residues on active receptor:Blacks binding of downstream SH2 domain containing antimotion protein/preventing pathway

•     test by mutating SH2 binding domains                        antimation
• -> SOCS Box 
•     recruits E3
•     ubiquitin ligase components

• -> RTK dimerization -> autophosporilates +    cross phosphorilation of tyrosine residues->
• -> SH2 domains containing proteins bind to phosphorilate typrosine residues in receptor
• -> SH3 domain containing proteins bind to SH3 domain on SH2 domains proteins