G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors. Upon ligand binding, GPCRs activate intracellular G proteins that propagate signals via second messenger molecules. There are three major families of G proteins - Gs stimulates adenylate cyclase and increases cAMP, Gi inhibits adenylate cyclase and decreases cAMP, and Gq activates phospholipase C and increases intracellular calcium. Receptor tyrosine kinases (RTKs) activate intracellular signaling pathways through autophosphorylation upon ligand binding, which recruits signaling proteins containing SH2 domains. Major RTK pathways include Ras-MAPK, which regulates cell growth and proliferation.

1. CELLULAR ASPECTS

2. Receptors
G-protein coupled receptors
 Ion Channel Receptors
Receptor-enzymes
Cytosolic-nuclear receptors

3. G-protein coupled receptors

4. G-protein coupled receptor: GPCR
Examples:
Adrenergic receptors
Muscarinic ACh receptors
GABAB receptors
Metabotropic Glutamate receptors
Dopamine receptors
Metabotropic Serotonin receptors

5. G-protein coupled receptor: GPCR
More examples—hormones:
Angiotensin receptor
Bradykinin receptor
Thrombin receptor
FSH receptor
LH receptor
TSH receptor
ACTH receptor

6. GPCR structure
A single subunit with
7 transmembrane segments
out
in

7. A depiction of how GPCRs activate signaling

8. Ligand + GPCR GPCR* GPCR* abg
a + bg
signaling
abg Heterotrimeric GTP-binding protein (G protein)
GPCR G-protein coupled receptor
signalin
g
Another depiction of how GPCRs activate signaling

9. Heterotrimeric GTP-binding proteins cycle between
GTP- and GDP-bound state
Binding of ligand to G-protein coupled receptor facilitates
exchange of GTP for GDP, a and bg dissociate
a
b g
GDP
a b g
GTP
INACTIVE ACTIVE

10. Heterotrimeric G proteins
•Activated by binding of ligand to 7-transmembrane receptor
(G-protein coupled receptor)
•Ligand binding causes dissociation of a and bg subunits
•Dissociation allows GDP to exchange for GTP
•GTP binding causes conformational change, a subunit can
now interact with effector (e.g. AC)
•GTP is hydrolyzed to GDP, a subunit dissociates from effector
•Signal is terminated

11. Adenylate cyclase
Membrane protein
makes cAMP from ATP
ATP cAMP

12. Three major families of G-proteins
Gs couples to Adenylate Cyclase
stimulates AC activity
increases cAMP
activates Protein Kinase A
Gi couples to Adenylate Cyclase
inhibits AC activity
decreases cAMP
inhibits Protein Kinase A
Gq couples to Phospholipase C
increases diacylgyclerol
(DAG)
increases IP3
increases intracellular Ca2+

13. Gs
Gi
Gq
b-adrenergic receptor
ACTH receptor
FSH receptor
a2-adrenergic receptor
M2 muscarinic receptor
a1-adrenergic receptor
M1, M3 muscarinic receptors
Angiotensin receptor
cAMP
PKA activity
cAMP
PKA activity
PLC activity
DAG, IP3
Ca2+
PKC activity
G-protein Receptor examples Signaling pathway

14. cAMP-dependent stimulation of glucose liberation from glycogen

15. bAR
a
b g
GTP
AC
PKA
cAMP-dependent stimulation of cardiac muscle contraction
Ca2+
Ca2+
Ryanodine
Receptor
Sarcoplasmic reticulum
bAR = b-adrenergic receptor

16. cAMP is constantly inactivated by
phosphodiesterase
(active) (inactive)
Theophylline blocks the phosphodiesterase

17. Phospholipase C activation produces Diacylglycerol and IP3
PI 4,5 biphosphate (PIP2)
Plasma membrane lipid
plasma
membrane
Diacylglycerol
(DAG)
Inositol
1,4,5-triphosphate
(IP3)
cytosolic messenger
plasma membrane

18. a1-adrenergic receptors acting on vascular smooth muscle:
PLC IP3 Ca2+ calmodulin myosin light chain kinase
Myosin-P contraction
An example of the Gq signaling pathway

19. IP3 receptors: intracellular calcium channels

20. Controlling intracellular calcium levels
IP3
receptors
Ca2+
Intracellular
Ca2+
levels are
<< micromolar

21. Guanylate cyclase receptors

22. Guanylate Cyclase Receptors
Two Types:
1. Transmembrane Guanylate Cyclase Receptor
activated by peptide hormones
2. Soluble Guanylate Cyclase Receptor
activated by nitric oxide (NO)
target for anti-angina drugs
nitroglycerin
Atrial natriuretic peptide/factor ANP

23. Transmembrane Guanylate Cyclase Receptor
3 domains
Hormone binding
Transmembrane
Intracellular GC
domain with
catalytic activity
Single transmembrane receptor—similar to tyrosine kinase receptor
(binds ANP)

24. Signal transduction initiated by binding of ANP to
Transmembrane Guanylate Cyclase Receptor
1. Atrial natriuretic peptide binds to receptor, causes
conformational change
2. Guanylate cyclase is activated
3. cGMP is generated
4. cGMP dependent protein kinase is activated
5. proteins are phosphorylated

25. Smooth muscle cell contraction
cGMP-dependent protein kinase (PKG) phosphorylates many
proteins that modulate muscle contraction
Ca2+ channels
K+ channels
IP3 receptors
Myosin light chain
phosphatase
NPR-A ANP receptor

26. Soluble Guanylate Cyclase Receptor
(binds NO)
NO, a gas, can
cross cell membranes
Interacts with heme
group on GC
inside the cell

27. Mechanism of activation of Guanylate Cyclase by NO
NOS nitric oxide synthase
catalyzes oxidation of arginine to produce citrilline + NO gas

28. Signal transduction initiated by generation of NO
1. Nitric oxide synthase (NOS) is activated in generator cell
2. Nitric oxide is formed by NOS from arginine
3. Gaseous nitric oxide diffuses out of generator cell and
into target cell
4. Nitric oxide reacts with iron-containing heme group on
Guanylate Cyclase
5. Activated Guanylate Cyclase converts GTP to cyclic GMP
6. cGMP binds to cGMP-dependent protein kinase (PKG)
7. proteins are phosphorylated by PKG

29. Mechanisms by which NO induces relaxation of smooth muscle
Smooth muscle contractionNO activates guanylate cyclase
cGMP is produced
cGMP dependent protein
kinase is activated
Proteins that regulate
contraction are
phosphorylated
IP3 R
MLCP
Ca2+
chann Ch
K+
K+
chann
PKG

30. Mechanisms by which NO induces relaxation of smooth muscle
(review)
NO Guanylate Cyclase cGMP PKG
IP3R K+ Chann Ca2+ chann Myosin light chain phosphatase

32. CELL
GROWTH
P
ras
MAPK MAPK P
phosphorlyates multiple
target proteins
PDGF
dimerize &
phosphorylate each
other
Mechanism of signal transduction

33. Enzyme-linked Cell Surface Receptors
 Receptor Tyrosine kinases: phosphorylate specific tyrosines
 Tyrosine kinase associated receptors: associate with
intracellular proteins that have tyrosine kinase activity.
 Receptorlike tyrosine phosphatases: remove phosphate
group
 Receptor Serine/ Threonine kinases: phosphorylate specific
Serine/ Threonine
 Receptor guanylyl cyclases: directly catalyzes the production
of cGMP
 Histidine kinase associated receptors: kinase phoshorylates
itself on histidine and then transfers the phosphate to a second
intracellular signaling protein.

34. Receptor Tyrosine Kinases (RTKs)
 Intrinsic tyrosine kinase activity
 Soluble or membrane-bound ligands:
 Nerve growth factor, NGF
 Platelet-derived growth factor, PDGF
 Fibroblast growth factor, EGF
 Epidermal growt factor, EGF
 Insulin
 Downstream pathway activation:
 Ras-MAP kinase pathway

35. TYROSINE KINASE RECEPTORS
• these receptors traverse the membrane only once
• respond exclusively to protein stimuli
– cytokines
– mitogenic growth factors:
• platelet derived growth factor
• epidermal growth factor

36.  Functions include:
 Cell proliferation, differentiation
 Cell survival
 Cellular metabolism
 Some RTKs have been discovered in cancer research
 Her2, constitutively active form in breast cancer
 EGF-R overexpression in breast cancer
 Other RTKs have been uncovered in studies of
developmental mutations that block differentiation

37. Outline
 Activated RTKs transmit signal to Ras protein
 Ras transduces signal to downstream serine-threonine
kinases
 Ultimate activation of MAP kinase
 Activation of transcription factors

38. Ligand binding to RTKs
 Most RTKs are monomeric
 ligand binding to EC domain induces dimerization
 FGF binds to heparan sulfate enhancing its binding to
receptor: dimeric receptor-ligand complex
 Some ligands are dimeric: direct dimerization of receptors
 Insulin receptors occur naturally as a dimer
 Activation is due to the conformational change of the
receptor upon ligand binding

39. Substrate + ATP Substrate-P + ADP
Protein Tyrosine Kinase
Protein Tyrosine Phosphatase
(PTP)

40. Tyrosine Protein Phosphorylation
• Eukaryotic cells coordinate functions through environmental signals -
soluble factors, extracellular matrix, neighboring cells.
• Membrane receptors receive these cues and transduce signals into the
cell for appropriate response.
• Tyrosine kinase signalling is the major mechanism for receptor signal
transduction.
• Tyrosine protein phosphorylation is rare (1%) relative to
serine/threonine phosphorylation.
• TK pathways mediate cell growth, differentiation, host defense, and
metabolic regulation.
• Protein tyrosine phosphorylation is the net effect of protein tyrosine
kinases (TKs) and protein tyrosine phosphatases (PTPs).

41. Protein Tyrosine Kinases (TKs)
Receptor tyrosine kinases (RTK)
– insulin receptor
– EGF receptor
– PDGF receptor
– TrkA
Non-receptor tyrosine kinases (NRTK)
– c-Src
– Janus kinases (Jak)
– Csk (C-terminal src kinase)
– Focal adhesion kinase (FAK)

42. TABLE 15–4 Some Signaling Proteins That Act Via Receptor Tyrosine Kinases
SIGNALING LIGAND RECEPTORS SOME RESPONSES
Epidermal growth factor (EGF) EGF receptor stimulates proliferation of various cell
types
Insulin insulin receptor stimulates carbohydrate utilization and
protein synthesis
Insulin-like growth factors IGF receptor-1 stimulate cell growth and survival
(IGF-1 and IGF-2)
Nerve growth factor (NGF) Trk A stimulates survival and growth of some
neurons
Platelet-derived growth factors PDGF receptors stimulate survival, growth, and
proliferation of various cell types
Macrophage-colony-stimulating M-CSF receptor stimulates monocyte/macrophage
factor (M-CSF) proliferation and differentiation
Fibroblast growth factors FGF receptors stimulate proliferation of various cell (FGF-
(FGF1 to FGF-24) (FGF-R1–FGFR4) types; inhibit differentiation of some
precursor cells; inductive signals in
development
Vascular endothelial growth VEGF receptor stimulates angiogenesis
factor (VEGF)
Ephrins (A and B types) Eph receptors (A and B) stimulate angiogenesis; guide cell and
axon migration

43. Signaling from tyrosine kinase receptors
• Ligand induced dimerization
• Autophosphorylation
• Phosphorylation in the catalytic domain increase
the kinase activity
• Phosphorylation outside the catalytic domain
creates specific binding for other proteins.
• Autophosphorylated receptors bind to
signaling proteins that have SH2
(phosphotyrosine residues) domains

44. Consequences of receptor dimerization
 Kinase in one subunit P* one or more tyrosine residues
on the other
 Binding of ATP (insulin-R) or protein substrates (FGF-R)
 Enhanced kinase activity: P* of other sites on the receptor
 P*-tyrosine residues become docking sites for adapter
proteins
 Small proteins with SH2, PTB and SH3 domains, but
without intrinsic enzymatic or signaling activities
 Coupling activated RTKs to components of signaling
pathways such as Ras

46. Channel Families
 Voltage-gated
 Extracellular ligand-gated
 Intracellular ligand-gated
 Inward rectifier
 Intercellular
 Other

47. Typical Ion Channels with Known Structure:
K+ channel (KCSA)
Types of ion channels:
 Simple pores (GA, GAP junctions)
 Substrate gated channels (Nicotinic receptor)
 Voltage-gated channels (K-channels)
 Pumps (ATP-synthase, K+,Na+-ATPase)

48. What are the Biochemical Changes that Lead to
Channel Gating (Opening or Closing)?
Gating involves some type of conformational change in the
protein, but other than that there are few definitive
answers to the question.
However, there are several general proposed models for
gating.

49. Types of Biochemical Mechanisms that
Open and Close Channels
 Conformational change occurs in a discrete area of the
channel, leading to it opening.
 The entire channel changes conformation (e.g., electrical
synapses).
 Ball-and-chain – type mechanism.
 Nt or hormone binding causes the channel to open.

50. Types of Biochemical Mechanisms that
Open and Close Channels (Cont’d)
 Nt or hormone binding to receptor causes a 2nd messenger
to activate a protein kinase that phosphorylates a channel
and thus opens it.
 Changes in membrane potential.
 Membrane deformation (e.g., mechanical pressure).
 Selectivity by charge (i.e., positively lined pore allows
anions through; negatively lined pore allows cations
through).

51. Extracellular ligand-gated
 nicotinic ACh (muscle): a2bg (embryonic), a2b
(adult)
 nicotinic ACh (neuronal): a(2-10), b(2-4)
 glutamate: NMDA, kainate, AMPA
 P2X (ATP)
 5-HT3
 GABAA: a(1-6), b(1-4), g (1-4), , , (1-3)
 Glycine

52. Intracellular ligand-gated leukotriene C4-gated
Ca2+
 ryanodine receptor
Ca2+
 IP3-gated Ca2+
 IP4-gated Ca2+
 Ca2+-gated K+
 Ca2+-gated non-
selective cation
• Ca2+-gated Cl–
• cAMP cation
• cGMP cation
• cAMP chloride
• ATP Cl–
• volume-regulated Cl–
• arachidonic acid-
activated K+
• Na+-gated K+

53. Gated Ion Channels
 Another type of membrane transport
 Pores in the membrane that open and close in a
regulated manner and allow passage of ions
-“Dispose” of the gradients
 Passive transporters
-Ions flow from high to low concentration
-No energy is used
-If there is no gradient ions will not flow

54. Gated Ion Channels
 Small highly selective pores in the cell membrane
 Move ions or H2O
 Fast rate of transport 107 ions x s-1
 Transport is always down the gradient
 Cannot be coupled to an energy source

55. Ion channels are everywhere
 Channels are present in almost every cell
 Functions
-Transport of ions and H2O
-Regulation of electrical
potential across the
membrane
-Signaling

56. Gating mechanisms
 Two discrete states ;open (conducting) closed
(nonconducting)
 Some channels have also inactivated state (open but
nonconducting)
 Part of the channel structure or external particle
blocks otherwise open channel

58. What gates ion channels?
 Non gated - always open
 Gated
􀁺 Voltage across the cell membrane
􀁺 Ligand
􀁺 Mechanical stimulus, heat (thermal fluctuations)

59. Gating mechanisms
 Conformational changes in channel protein are
responsible for opening and closing of the pore
-3D conformational shape is determined by atomic,
electric, and hydrophobic forces
 Energy to switch the channel protein from one
conformational shape to another comes from the
gating source

60. Ligand gated channels
 Glutamate receptors
 Nicotinic acetylcholine receptor
 Vanilloid receptor family (TRPV)
= Neurotransmitter Ion Flow = Current

61. Ligand gated ion channels
 Gated by ligands present outside of the cell
 In fact they are receptors
 All of them are nonselective cation channels
 Mediate effects of neurotransmitters

63. Nuclear Receptors
1. Proteins interact with steroids and other
hormones that diffuse through the cell
membrane.
2. Form hormone-receptor complexes that function
as activators by binding to enhancers 
hormone response elements.
3. Sex hormones: estrogens and androgens;
glucocorticoids, cortisol, vitamin D  Ca2+
metabolism; thyroid hormone, retinoic acid 
developmental factors.

64. 1. The majority of nuclear receptors bind to respective
enhancer elements and repress transcription.
- In the presence of hormone, they form R-H
complexes in the nucleus and function as activators
by binding to the same enhancers.
- Act as repressor or enhancer, depending on the
physiological signals.
- thus, the response element serves as either
enhancer or silencer.

65. Responses to hydrophobic hormones are mediated by
intracellular receptors
Transcription
Translation
Cytoplasm
Nucleus
Nuclear
envelope
Plasma
membrane Lipophilic hormone carried in
blood
Hormone binds intracellular
receptor inducing receptor
dimerization and activation
Complex is imported into
nucleus
Binds to “hormone response
element” to regulate gene
expression
Intracellular
receptor
Promoter Target gene“Hormone
response
element”
Target
cell
Lipophillic
Hormone

66. 2. The glucocorticoid (nuclear) receptor is found in the
cytoplasm

67. Glucocorticoid Action
1. GR exists in an inactive form in the cytoplasm 
complexed with heat shock protein 90 (hsp90).
2. Glucocorticoid (G) diffuses across cell membrane
and enters cytoplasm
3. G binds to GR  changes conformation 
dissociates from hsp90
4.  exposes a nuclear localization signal (stretch of
aas) on GR.
5. G-GR (hormone-receptor complex, HR) enters
nucleus, dimerizes with another HR.

68. 6. HR dimer binds to enhancer/hormone-response
element upstream of hormone activated gene.
7. Binding of HR dimer to enhancer  activates
transcription.
8. Most contain 2 zinc fingers (1) controls DNA
binding, (2) controls dimerization
Critical residues for discriminating
between GRE and ERE lie at the base
of the first finger
-GRE = glucocorticoid responsive element
/enhancer (sequence); ERE = estrogen

69. Specificity of DNA binding
dimerization