12 G Protein/Wnt kinase pathway|Tulane

Signaling at the cell surface

Chapter 15, pages 687-712
Chapter 16, pages 752-757

GPCR-Related Researchers Awarded Nobel Prize

• 1967 Ragnar Granit, Haldan Keffer Hartline and George Waki – physiological and chemical visual processes in the eye.
• 1970 Bernard Katz, Ulf von Euler and Julius Axelrod – neurotransmitters in the nerve terminals and the mechanism for their storage, release and inactivation.
• 1971 Earl Wilbur Sutherland, Jr. – discovered cyclic AMP as the second messenger for mediating the action of hormones.
• 1988 James W. Black – discovery of propranolol and cimetidine, two clinical drugs that block the action of the b-adrenergic receptor and the H2 histamine receptor, respectively
• 1994 Alfred G. Gilman and Martin Rodbell – discovery of G-proteins and their role in signal transduction
• 2000 Arvid Carlsson - dopamine
• 2004 Richard Axel and Linda B. Buck – odorant receptors
• 2012 Robert J. Lefkowitz and Brian K. Kobilka - identification, purification, cloning and determining crystal structure of the adrenergic receptors

General Elements of GPCR Systems

• Accepter
• Trimeric G_s protein
• Effector

• contains 7 membrane-spanning domains
• coupled trimeric G protein - functions as a switch (active/inactive)
• A membrane bound effector protein
• Most involve second messengers
• Feedback regulation and desensitization of the signaling pathway

Most receptors are G protein-coupled receptors (GPCRs)
• Contains 7 membrane-spanning domains
• A coupled trimeric G protein which functions as a switch
• A membrane bound effector protein
• Most involve second messengers
• Feedback regulation and desensitization of the signaling pathway

General structure of GPCRs
The same orientation in the membrane
7 transmembrane
α-helical regions
4 extracellular segment
4 cytosolic segments

Rhodopsin structure
β2-Adrenergic Receptor

Dendrogram of human GPCR superfamily

crystal structures
solved by Nov 2012

A coupled trimeric G protein which functions as a switch

G protein

G Protein + GTP → Conformation changed G Protein + GDP + Pi
G Protein: on/off state

Heterotrimeric G proteins vary in composition

3 subunit: G_α, G_β, G_γ

• 23 a isoforms (39-46 kDa proteins)
• contains the GTP/GDP binding site
• is responsible for identity
• membrane anchored through prenylation
• 6 b isoforms (35 - 36 kDa proteins) and 12 g isoforms (8 kDa proteins)
• are functionally identical or very similar, interchangeable in vitro;
• most of them are ubiquitously expressed;
• membrane anchored through prenylation of Gβ.

A membrane bound effector protein

One side is activate, another side has a inactivate proteins

• Most involve second messengers
• Feedback regulation and desensitization of the signaling pathway

General Elements of GPCR Systems

Operational model:

Hormone bind to the Active receptor
G protein bind to the Active receptor
GDP released
G Protein + GTP: α subunit sepratede with other two
α subunit bind to the active effector, GTP → GDP

Which of the following mutations to the Ga protein could render a G protein-coupled receptor signaling pathway constitutively active?
A) Ga cannot bind Gbg
B) Ga cannot hydrolyze GTP
C) Ga protein cannot bind GDP
D) Ga protein cannot release GDP
E) Ga protein cannot bind GTP

Which of the following mutations to the Ga protein could render a G
protein-coupled receptor signaling pathway constitutively active?
A) Ga cannot bind Gbg
B) Ga cannot hydrolyze GTP
C) Ga protein cannot bind GDP
D) Ga protein cannot release GDP
E) Ga protein cannot bind GTP
A Ga subunit that cannot hydrolyze
GTP would remain bound to its
downstream effector, keeping it
constitutively active. All of the other
mutations would render the signaling
pathway nonfunctional.

Heterotrimeric G protein families (based on a subunit)

GPCRs mediated Regulation of Ion Channels

Accepter + G protein + Ion Channel
After activated, β and γ bind to the ion channel

• Neurotransmitter receptors (Na and K channels)- Nerve impulses essential for sensory perception
• Acetylcholine receptors (K channel)- Hyperpolarizes membrane, reduces frequency of heart muscle contraction

• Human retina contains 2 types of photoreceptors - cones and rods
• Cone photoreceptors contain the GPCRs for color
• Rod photoreceptor cells contain GPCR (rhodopsin) for non-color light
• Rhodopsin is localized in the thousands of flattened membrane disks in the outer segment of rod cells

The light-triggered step in vision.

Rhodopsin = opsin (7 transmembrane protein) + 11-cis-retinal (light absorbing pigment)

Light-activated rhodopsin pathway and the closing of ion channels in rod cells

Ligand = photon (light)
Transducer = Gat (transducin)
Effector = cGMP phosphodiesterase, reduces cGMP in the cell
*reduced cGMP causes cGMP-gated ion channel to close, membrane becomes hyperpolarized, less neurotransmitter released, brain sees this as ‘light’

Inhibition of rhodopsin signaling by rhodopsin kinase

• Light activated rhodopsin is substrate for rhodopsin kinase
• The extend of rhodopsin phosphorylation proportional to the amount of time each rhodopsin spend in the light
• Arreastin binds to completely phosphorylated Rhodopsin – no activation

Mechanism for re-setting visual cycle

1. Hydrolysis of GTP on α-subunit – accelerated by GAP
2. Re-synthesis of cGMP by guanyl cyclase is stimulated by lower Ca++ levels
3. Phosphorylation of cytoplasmic loop of rhodopsin (opsin) allowing β-arrestin to bind
4. Phosphatases remove phosphates from cytoplasmic loop of rhodopsin resensitizes

Most involve second messengers

Secondary messenger

• cAMP
• cGMP
• DAG
• IP₃

GPCRs activate or inhibit adenylyl cyclase

Binding of Gsa-GTP to adenylyl cyclase activates the enzyme, whereas binding of Gia -GTP inhibits adenylyl cyclase. The Gbg subunit in both stimulatory and inhibitory G proteins is identical; the Ga subunits and the receptors differ.

• Gsa stimulates adenylyl cyclase to increase cAMP
• Virtually all the diverse effects of cAMP are mediated through PKA
• The resulting cellular response depends on the particular PKA isoform and the PKA substrates expressed by the cell

PKA Links GPCRs to transcription

CREB = CRE-binding protein, found in the nucleus, phosphorylated and activated by PKA
CRE = cAMP responsive element

Table 15-2

mAKAP anchors both PKA and PDE to the nuclear membrane, maintaining them in a negative feedback loop

1. Basal PDE activity: Resting state
2. Increased cAMP: PKA activation
3. PDE phosphorylation and activation; reduction in cAMP level

mAKAP - A kinase-associated protein kinase; anchoring
PDE - cAMP phosphodiesterase
PKA - protein kinase A

Synthesis of 2^nd messengers DAG and IP3

IP3/DAG pathway and the elevation of cytosolic Ca²⁺

Provides close local control of the cAMP level
30
cytosol
membrane
Actin remodeling
Endocytosis
Vesicle fusion
2nd messengers

table 15-3

Feedback regulation and desensitization of the signaling pathway

Inactivate receptor + G Protein + Inactive effector

Mechanisms to attenuate signaling by GPCRs

• Removal of stimulus - reuptake, degradation
• Affinity of receptor for ligand decreases when GDP bound G- protein becomes replaced with GTP
• GTP is quickly hydrolyzed, inactivating Ga (e.g. reversing the activation of adenylyl cyclase & production of cAMP)
• Removal of 2nd messenger (e.g. cAMP phosphodiesterase hydrolyzes cAMP to 5’-AMP, terminating the cellular response)
• Receptor desensitization
• Receptor endocytosis - recycling or degradation

GPCR desensitization

• Feedback suppression
• Heterologous desensitization
• Homologous desensitization

• Feedback suppression
  The end product of a pathway blocks an early step in the pathway
  Example: activation of receptor Leads to activation of PKA, PKA phosphorylates the same receptor, reducing its ability to activate adenylyl cyclase
• Homologous Desensitization
  • Agonist-dependent phosphorylation of receptors mediated by G protein-coupled receptor kinases (GRKs)
  • Receptor interaction with intracellular proteins called arrestins
  • Arrestin binding sterically precludes coupling between the receptor and heterotrimeric G proteins
  • Termination of signaling by effector

G protein coupled receptor kinases (GRKs)

N Catalytic Domain
0 50 100
amino acids
amino-terminal domain
(receptor-binding?)
autophosphorylation
region
Variable region
(membrane targeting)
C

• Rhodopsin kinase or visual (GRK1 and GRK7)
• β-adrenergic receptor kinases (GRK2/GRK3)
• GRK4 (GRK4, GRK5 and GRK6)

Heterologous Desensitization

Activation of one type of receptor can activate signal transduction leading to the desensitization of a different receptor (agonist-independent phosphorylation)
Luttrell L M , and Lefkowitz R J J Cell Sci 2002;115:455-465
©2002 by The Company of Biologists Ltd

β-arrestins can act as adapters

Receptor-bound b-arrestins also act as adapter proteins, binding to components of the clathrin endocytic machinery and other signaling molecules.

β-arrestin-induced signaling

• RhoA-dependent stress fiber formation
• inhibition of nuclear factor κB (NF-κB)- targeted gene expression through IκB stabilization
• Protein phosphatase 2A (PP2A)- mediated dephosphorylation of Akt, which leads to the activation of glycogen synthase kinase 3 (GSK3)
• Extracellular signal-regulated kinase (ERK)-dependent induction of protein translation and antiapoptosis
• Phosphatidylinositol 3-kinase (PI3K)- mediated phospholipase A2 (PLA2) induction and increased vasodilation through GPR109A activation
• Kif3A-dependent relocalization and activation of the protein Smoothened (Smo) in the primary cilium (21).
  44
  Phosphorylated (desensitized) receptors are constantly being
  resensitized owing to dephosphorylation by constitutive
  phosphatases.
  All involve phosphorylation of GPCR
  GPCR desensitization
  • Feedback suppression
  • Heterologous desensitization
  • Homologous desensitization
  Which of the following steps in an intracellular signaling pathway amplifies
  the signal?
  A) binding of ligand to receptor

• B) synthesis of a secondary messenger
• C) activation of a protein kinase
  D) receptor desensitization
• E) receptor activation of a G protein
  In B, C, and E, each signaling molecule produces/activates more than one of the next signaling molecule.

GPCR Signaling Diversity

Oligomerization of GPCRs can confer unique pharmacological and functional profiles to a receptor, including affinity for specific peptide ligands and coupling to novel G proteins.
Both homo- and hetero-oligomeric receptor complexes may have the ability to couple to more than one G protein, depending on their cellular environment, thereby conferring the ability to mediate different intracellular responses to the same ligand.

Disruption of G protein signaling can cause disease

Cholera

• Cholera toxin catalyzes covalent modification of Gsa. ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gsa.
• ADP-ribosylation prevents Gsa from hydrolyzing GTP. Thus G
sa becomes permanently activated.
• Resulting excessive rise in intracellular cAMP leads to loss of electrolytes and water into the intestinal lumen, producing watery diarrhea.

Whooping Cough Disease

Colonization of tracheal epithelial cells by Bordetella pertussis
Pertussis toxin catalyzes ADP-ribosylation at a cysteine residue of Gia, making the inhibitory Ga incapable of exchanging GDP for GTP. Thus the inhibitory pathway is blocked
Resulting increase in cAMP in epithelial cells of the airways promotes loss of fluids and electrolytes and mucus secretion.

These toxins can be useful tools in the lab

• used to determine whether a particular agonist of a GPCR invokes activation of members of the Gia or Gsa family
• If the GPCR mediates its effects through Giawe should see an increase in activity (cAMP) when we apply the pertusis toxin compared to control. If the GPCR mediates its effects through Gsa we should see a significant increase in activity (cAMP) when we apply the cholera toxin.

Many Human Viruses Encode GPCRs

Wnt and Hedgehog signaling Nonconventional 7-TM Receptors

Frizzled family receptors

Schulte. Pharmacological Reviews. 2010; 62:633

Wnt Proteins

• Large family of secreted molecules (350 to 400 aa) highly conserved across species

• 2 members discovered:

• Drosophila Wingless
• Mouse int-1

• Three Wnt signaling pathways:

• canonical Wnt pathway (operates through b-catenin)
• noncanonical planar cell polarity pathway
• noncanonical Wnt/calcium pathway

Control processes during development, stem cell maintenance and homeostasis
Aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer

Canonical Wnt signaling Activation of b-catenin

MacDonald et al. Developmental Cell 17, July 21, 2009

APC – adenomatosis polyposis coli
CK – casein kinase
GSK3 – glycogen synthase kinase 3b
Gro – groucho

Noncanonical Wnt signaling

Niehrs. Nature Reviews. 2012; 13:767
60
MacDonald et al. Developmental Cell 17, July 21, 2009
61
Cancer
Inflammatory bowel
disease
Parkinsons
Diseases of bone
Obesity

Wnt related diseases

Hedgehog signaling pathway

• Discovered in Drosophila and is conserved in vertebrates (including humans)
• Involved in cell growth and differentiation during embryonic development

• ensuring that tissues reach their correct size and location, maintaining tissue polarity and cellular content
• regulating hair follicle and sebaceous gland development In
  the skin

• Germline mutations in of the results in a number of developmental abnormalities
• Remains inactive in most adult tissues

Hedgehog signaling

Abnormal Hedgehog pathway signaling in the pathogenesis of certain types of cancers

Two different mechanisms drive abnormal Hedgehog pathway signalling in different types of cancer:

1. Ligand-independent signalling driven by mutations (e.g. BCC and medulloblastoma) Mutations in key pathway regulators (e.g. PTCH or SMO) cause SMO to be in a constitutively active state
2. Ligand-dependent signalling driven by overexpression of Hh ligand by tumour cells (e.g. ovarian cancer, colorectal cancer, pancreatic cancer) Dorsam and Gutkind Nature Reviews Cancer 7, 79–94 (February 2007) | doi:10.1038/nrc2069

Please read
Molecular Cell Biology
Chapter 15, pages 687-712
Chapter 16, pages 752-757
Thank you
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Operational model for ligand-induced activation of effector proteins
associated with GPCRs
http://www.youtube.com/watch?v=V_0EcUr_txk