L26 Cell Signalling II

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

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

  • Rs have 2functions
  1. binding a h w/ high affinity
  2. 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)

  1. G prot-coupled Rs
  2. a) Rs tyrosine kinase
  3. b) Kinase activates TF, altering gene exp
  4. Rs guanylyl cyclase
  5. Gated ion channel
  6. adhesion Rs (intergrin)
  7. 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

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)

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 can be located

  1. in the CYTOSOL (eg. estrogen Rs) & are transported to the nuc after the lgd is bound
  2. 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

  1. H binding its spec Rs
  2. H-Rs complex bindng spec regions of DNA, ultimately leading to upregulation of spec prots

Steroid Hs - mode of action... Loking @ GENE PROMOTERS

  1. Trc reqs the ezm : RNA pol
  2. 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