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L26 Cell Signalling II (Nuclear Rs
bring abt an effect in nuc…
L26 Cell Signalling II
Studying Rs/Lgd Specificity
- Rs: bind c spec to the Hormone
- binding specificity of a Rs for a spec lgd = a measure of ability of the Rs to distinguish between closely related subs
- eg. insulin binds w/ high spec to spec molecs
~ IGF-1 & IGF-2 but not ther hormones
Binding spec
- crucial for good health
- e,g EPO is quite sim to other cytokines GH, PRO, ILs etc but the EPO-R (bone marrow) only binds EPO to make more red bld (c)s, thankfully, otherwise there would be chaos
Signalling Activity of Rs
- binding a h w/ high affinity
- invoking a signal inside the (c) to initiate a biological response (in the TC)
- h + Rs : in absence of h the Rs doesn't convey any info = INACTIVE
~ not conveying the sig to the (c)
Specificity
- sig molec fits binding site on its complementary Rs: other sigs don't fit
- analogy: having a key
when Rs prot encounters the h, it binds the h
- ∆s in Rs conform allow ∆s in interactions w/ other molecs INSIDE (c) = ACTIVE
- ∆s: often dictated thru alterations in shape (conformation)
Rs binding studies
- Rs binding of a hormone is spec, ∴ Rs is a HIGH-AFFINITY binding prot
~ (similarities can be drawn w/ ezms & how they can bin their substrates thru spec interactions)
- the interactions of a h and Rs are HIGHLY-spec
~ involve non-cov interactions- ionic & van der Waals
- Binding induces CONFORMATIONAL CHANGES in the Rs
~ detected by INTRACELLULAR MOLECS
Filter Assay
- Rationale:
~ Equilibrium binding of labeled lgd w/the Rs
~ R + L ⇌ RL
~ bound complex becomes radioactive
~ free Rs remains non-radioactive
~ free lgd can pass thru filter
~ complex cant pass bc prot binds to the filter
- Steps:
~ isolate membs
~ add lgd to membs
~ pass thru a filter
~ wash off unbound lgd
~ measure bound radioactively, which is proportional to [COMPLEX]
- can calculate how many Rs are on (c) surface
Non-spec binding
- Problem: hydrophobic lgd are nonspecifically soaked into memb
- Solution:
~ measure total binding
~ measure nonspec binding (NSB) in the absence of Rs
~ substract NSB from Total to get specific binding
~ analyze spec binding data
Data
- binding become saturated: bc only have finite #Rs on (C) surface & once all occupied => no more further binding
- te determine spec binding = Total - nonspec
Data Analysis
- slope = -1/Kd
- Bmax= maximal binding
of h to Rs (#Rs)
- Bound h, [RL] = conc of h @ which half maximal binding ocurs = Kd (units =M)
Signalling thru the memb (6 diff types of transducers)
- G prot-coupled Rs
- a) Rs tyrosine kinase
- b) Kinase activates TF, altering gene exp
- Rs guanylyl cyclase
- Gated ion channel
- adhesion Rs (intergrin)
- Nuclear Rs
Nuclear Rs
- bring abt an effect in nuc
Simple comm pathways involving small hydrophobic signalling molecs
- sm, hydrphobic molecs can move across CM [using carrier prot]
~ ∴ the Rs to these signallng molecs are found INSIDE the (C) [cytoplasm or nuc]
- ie nuclear Rs signalling e.g. steroids
Signalling of nuc Rs
- steroid Hs: derived from cholesterol by stepwise removal of C atoms & hyrdoxylation
- Steroid Hs: synthesised in adrenal cortex (the glucocorticoids[binds to glucocorticoid Rs] & mineralocorticoids[role in electrolyste balance]) & also the gonads (for eostrogen, progestins, androgens)
~ [sex steroids = huge role in repro syst]
NR signalling molecs
- small & hphobic, lipid-sol
- can diffuse thru memb of TCs
- bind to intra(c) Rs
- all functions in same basic manner
- binding to the intra(c) Rs affects the (C)s ability to transcribe spec genes
- interact DIRECTLY w/ TFs they control
Luteinizing hormone
- = stimulus for androgen production
- B has the Rs & capable of binding h which brings abt a target response to tht (c)
- a #TCs including:
~ prostrate
~ bone
~ muscle
Target responses are varied:
- maintenance of functional male repro syst
~ & 2o male characs
- Anabolic action - skeletal growth
~& skeletal muscle growth
NRs superfam
~ All NR share common domain st
- Central DNA-binding domain: the part which contacts (binds) DNA
- Lgd binding domain (SPECIFICITY)- hormone dependent activation (or repression)
- Variable N terminal domains which may contain one or more activation domains - to alter gene Trc (exp)
Rs Activation
- Rs, in inactive states = bound to inh prots
~ eg. Estrogen Rs bound to heat shock prots (hsp90)
- Binding of lgd induces a conformational ∆ releasing the inh prot & binding a co-activator prot to induce gene Trc (co-repressors eg tamoxifen)
Rs can be located
- in the CYTOSOL (eg. estrogen Rs) & are transported to the nuc after the lgd is bound
- in the NUC ( thyroid & retinoid Rs)
NR Response Element
- the nt binding seq tht interact w/ NR = response element
- RE lie adj to the genes tht the h (lgd) regulates
- consist of 6bp (smtms inverted) repeated seqs separated by 3-5bps
- eg. only glucocorticoid complexes will bind to the glucocorticiod RE
- Despite similarity in RE, il i y specificity in biinding
Specificity
- binding of Rs is to spec seqs in the DNA
Trc
spec mRNA produced (when lgd interracts e/ the Rs
Trl
Spec prot is produced
2 lvls of spec in
- H binding its spec Rs
- H-Rs complex bindng spec regions of DNA, ultimately leading to upregulation of spec prots
Steroid Hs - mode of action... Loking @ GENE PROMOTERS
- Trc reqs the ezm : RNA pol
- Std H Rs must ∴be influencing RNA pol
- RE in the promoter reg: binds the Rs/H complex & allow for . std H to ⇈ gene Trc
- the Lgd/H Rs complex is a TF: translocate since the nuc recogs spec response in the gene, binds to tht reg and induces Trc
Signalling of NRs - Direct
- H + NRs → "Direct" signal transduction
- no other intermeds involved
- the Rs IS the TF
- the interaction of H w/ Rs directly induces gene Trc
- il y a ↓opportunity for regu by other factors & pathways (bc its direct)
- limited amplification of signal
Learning outcomes
- Understand tht Rs/ligand interaction is v spec
- Understand how Rs ligand studies are performed & be able to describe Kd & Bmax represent
- Understand the properties of small hydrophobic signalling molecs
- Describe the steroid hormone signal transduction mech w/ particular reference to regu of gene Trc