132 Endocrine Signaling

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13-2 Endocrine Signaling / Signal Transduction
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Cell Signaling

• The Insulin Receptor Cascades

Insulin Receptor


PI-3 kinase
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Cell Signaling

• The Insulin Receptor Cascades
• Ras
• ras gene - originally discovered as a viral
  oncogene
• Also part of the normal genome
• Many tumor cells contain a mutant ras
• Protein product Ras
• Small G-protein
• At inner surface of plasma membrane
• Lipid group embedded in inner leaflet
• Forms an important link to nucleus via mitogen
  activated protein kinases (MAPK)

4
Cell Signaling

• The Insulin Receptor Cascades
• MAPK
• Best characterized group extracellular
  signal-regulated kinase (ERK) family
• Act via either of two pathways
• RTKs such as the insulin cascade
• Also via G-protein coupled receptors
• ERK activation via coupling to
• Ras and
• Raf a protein serine/threonine kinase

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Cell Signaling

• The Insulin Receptor Cascades
• Ras Raf and MAPK

Insulin
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Cell Signaling

• The Insulin Receptor Cascades
• Ras Raf and MAPK

test



SOS (is a GEF)
Grb-2
GTP
GDP
SH2 domain
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Cell Signaling

• The Insulin Receptor Cascades
• Raf recruitment
• Raf then phosphorylates MAPK/ERK kinase known as
  MEK
• MEK has dual specificity phosphorylates
• Threonine
• Tyrosine
• These are separated by one aa residue
• Example ERK2 Thr 183 and Tyr 185
• Downstream effects of ERK - transcription

8
Cell Signaling

• The Insulin Receptor Cascades
• Mechanism of Ras activation
• Mediated by guanine nucleotide exchange factors
  (GEF)
• Stimulate release of bound GDP
• Exchange for GTP
• Activation of Ras-GTP terminated by GTP
  hydrolysis
• GTPase-activating proteins interact and
  accelerate GTP hydrolysis

9
Cell Signaling

• The Insulin Receptor Cascades
• Mechanism of Ras activation

10
Cell Signaling

• The Insulin Receptor Cascades
• Ras activation
• A well characterized GEF is Sos
• Sos is bound to Grb2
• Found in the cytosol
• Grb2 is an adaptor protein
• Has no other intrinsic activity
• Has SH2 domain high affinity for
  phospho-tyrosine (eg on IRS)
• Sos-Grb2 localized to the membrane via Ras

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Cell Signaling

• The Insulin Receptor Cascades
• Ras activation
• Sos-Grb2 localized to the membrane via Ras
• Sos stimulates GTP exchange activating Ras

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Cell Signaling

• The Insulin Receptor Cascades
• ERK activation
• As discussed Raf activates the MAPK cascade
• ERK translocates to the nucleus
• Regulates transcription factors by
  phosphorylation

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Cell Signaling

• The Insulin Receptor Cascades
• ERK activation
• Many growth factors invoke rapid transcriptional
  induction of immediate-early genes
• Mediated via serum response element
• Recognized by several factors including
• SRF
• Elk1

14
Cell Signaling

• The Leptin Receptor Cascades
• Cytokine superfamily receptors
• No catalytic domain
• Interact with nonreceptor protein-tyrosine
  kinases
• Src family
• JAK family

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Cell Signaling

• The Leptin Receptor Cascades
• JAK/STAT Pathway
• More immediate connection to transcription
• Directly affects transcription factor
  localization and function
• Key elements signal transducers and activators
  of transcription (STATs)
• Also other interactions via MAPK and PI3K

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Cell Signaling

• The Leptin Receptor Cascades
• JAK/STAT Pathway
• STATs contain SH2 domains
• Inactive in unstimulated cells
• Localized to cytoplasm
• Leptin binding to its receptor causes recruitment
  of STATs which bind to phosphotyrosine via SH2
• STATs are then phosphorylated by Janus Kinase
  (JAK)
• STATs then dimerise and are translocated to the
  nucleus stimulation of transcription

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Cell Signaling

• The Leptin Receptor Cascades
• JAK/STAT Pathway

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Cell Signaling

• The b-adrenoceptor Cascades
• Binding of a small messenger molecule
• Epinephrine/norepinephrine
• Interaction with the receptor causes a
  conformational change
• Activates G-protein binding sites in C-terminal
  domain
• G-protein interacts with another integral
  membrane protein (enzyme) adenylyl cyclase
• Downstream effects via cyclic-AMP

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Cell Signaling

• The b-adrenoceptor Cascades

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Cell Signaling

• The b-adrenoceptor Cascades

21
Cell Signaling

• The b-adrenoceptor Cascades
• Mechanism of G-protein signaling
• Cyclic AMP an important messenger for a number of
  hormone axes
• GTP essential for activation of the enzyme which
  generates cyclic AMP adenylyl cyclase

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Cell Signaling

• The b-adrenoceptor Cascades
• Mechanism of G-protein signaling

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Cell Signaling

• The b-adrenoceptor Cascades
• G-protein structure
• Three subunits a, b, g
• Heterotrimeric G-proteins
• a subunit binds guanine nucleotides regulatory
• When inactivated a is bound to GDP and the b and
  g subunits
• a and g subunits also attached to membrane via
  lipid moieties

24
Cell Signaling

• The b-adrenoceptor Cascades
• G-protein function
• Hormone binding induces a conformational change
  in the receptor
• C-terminal domain of receptor interacts with
  G-protein
• Release of bound GDP
• Exchange for GTP
• Activated a subunit dissociates
• a subunit activates adenylyl cyclase

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Cell Signaling

• The b-adrenoceptor Cascades
• G-protein function

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Cell Signaling

• G-protein diversity
• 20 different a subunits
• 6 b subunits
• 12 g subunits
• Different G-proteins associate with different
  receptors (c-terminal domain specificity)
• Specific intracellular targets
• b-adrenoceptor-linked G-protein Gs
• Stimulates adenylyl cyclase
• Other G-proteins inhibit adenylyl cyclase - Gi

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Cell Signaling

• The b-adrenoceptor Cascades
• Cyclic AMP pathway
• Formed from ATP via adenylyl cyclase action
• Degraded to AMP by phosphodiesterase

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Cell Signaling

• The b-adrenoceptor Cascades
• Cyclic AMP pathway
• Most effects of cAMP mediated via protein kinase
  A (PKA)
• cAMP binds to regulatory subunits of PKA
• Regulatory and catalytic subunits dissociate
• Catalytic subunits activated
• Phosphorylate serine residues on target proteins
• Good example of signal amplification

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Cell Signaling

• The b-adrenoceptor Cascades
• Cyclic AMP pathway
• Activation of protein kinase A

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Cell Signaling

• The b-adrenoceptor Cascades
• Cyclic AMP pathway
• Many cases activation of transcription of
  target genes containing cAMP response element
  (CRE)
• Signal carried by catalytic subunit of PKA
• Phosphorylates transcription factor CRE-binding
  protein (CREB) - dimerises
• Activation of cAMP-inducible genes

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Cell Signaling

• The b-adrenoceptor Cascades
• Cyclic AMP pathway

32
Cell Signaling

• Cyclic AMP pathway
• Important in proliferation, differentiation of
  many cell types
• Protein kinases
• Protein kinase A
• Not independent action
• Protein phosphorylation reversed by phosphatases
• Terminate responses following receptor activation
  of protein kinases

33
Cell Signaling

• Protein kinases
• Protein kinase A
• Level of phosphorylation of substrates finely
  controlled by balance of actions of the kinase
  and phosphatases

34
Cell Signaling

• The Insulin Receptor Cascades Interaction with
  the Leptin Pathway