Regulation of GPCR signaling

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Regulation of GPCR signaling
Seva Gurevich
Vsevolod.Gurevich_at_vanderbilt.edu
GPCR - G protein-coupled receptor Why would you
want to know anything about GPCRs Out of about
30,000 genes in human (or mouse) genome, 1,000
(gt3) encode GPCRs.
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(From Bockaert Pin (1999) EMBO J. 18,
1723-1729)
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STRUCTURE OF RHODOPSIN
A distorted barrel consisting of seven
transmembrane a-helices. Extracellular
N-terminus, cytoplasmic C-terminus.
Palczewski, Kumasaka, Hori, Behnke, Motoshima,
Fox, Le Trong, Teller, Okada, Stenkamp, Yamamoto,
Miyano (2000) Science 289, 739-745.
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BOVINE RHODOPSIN
Menon, Han, Sakmar (2001) Physiol. Rev. 81,
1659-1688.
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GPCR Signaling Cascade
light
Amplification
GPCR
GPCR
agonist
10-200
Gabg
Gabg
Gabg
GaGTP
Gbg
Effectors
Effectors
100-10,000
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The saga begins G protein-coupled receptor
kinases (GRKs) and arrestins come into
play. When? Why? And how?
GRK
light
GPCR
GPCR
GPCR
GPCR
agonist
P
P
P
P
P
P
Arrestin
Arrestin
Gabg
Gabg
Gabg
X
GaGTP
Gbg
Effectors
Effectors
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• Why do we think that the regulation of GPCR
  signaling is important?
• The knockout of GRK2 (the most ubiquitous GRK in
  mammals) is embryonic lethal in mice.
• Simultaneous knockout of both non-visual
  arrestins is embryonic lethal in mice.
• The knockout of the only non-visual arrestin that
  Drosophila has (kurtz) is also embryonic lethal.
• The absence of either visual arrestin or
  rhodopsin kinase results in light-dependent
  retinal degeneration in mice and flies (causes
  Oguchi disease, i.e., congenital night blindness,
  in humans).
• Defects in rhodopsin precluding its normal
  phosphorylation and arrestin-dependent signal
  shut off result in retinitis pigmentosa (retinal
  degeneration and blindness) in humans.

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What do we have Seven GRKs (termed GRK1 through
GRK7) and four arrestins. Visual arrestin
(arrestin1) and GRK1 in rod photoreceptors. Cone
arrestin (arrestin4) and GRKs 1 and 7 in cone
photoreceptors. Non-visual arrestins, arrestin2
(a.k.a. b-arrestin1) and arrestin3 (a.k.a.
b-arrestin2), and GRKs 2, 3, and 5 in virtually
every cell of the body (plus GRKs 4 and 6 in
some).
Virtually every cell has many (5-20?) different
GPCRS, at least two arrestins, and three or more
GRKs. GPCRs are integral membrane proteins
localized in the plasma membrane. GRKs and
arrestins are soluble cytoplasmic proteins. How
do they get to their GPCR targets?
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Recruitment of G protein-coupled receptor
kinases to the membrane

• Constitutive membrane association
• 1. Due to lipid modifications
• GRK1 - farnesylated
• GRK7 - geranylgeranylated
• GRK4 GRK6 - palmitoylated
• 2. Due to direct binding to phospholipids
• GRK5 - via positively charged domain near its
  C-terminus
• Reversible membrane association
• GRK2 GRK3 employ two mechanisms
• Interaction with released Gbg via plekstrin
  homology (PH) domain in their C-termini
• Interaction with Gaq via RGS domain in their
  N-termini.

Reviewed in Kohout Lefkowitz, Mol. Pharmacol.
63, 9-18 (2003)
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Q How does the GRK know when to phosphorylate
the receptor? A Activated receptor itself
activates GRK.
Palczewski, Buczylko, Kaplan, Polans, Crabb
(1991) J. Biol. Chem. 266, 12949-55.
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How does arrestin know when to bind?
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Sequential multi-site binding ensures arrestin
selectivity for P-Rh
Arrestin works as a molecular coincidence
detector, going onto high-affinity
receptor-binding state when both phosphate sensor
and active receptor sensor are simultaneously
engaged.
Gurevich, V.V., and Benovic, J.L. (1993) J. Biol.
Chem. 268, 11628-11638. Figure from Gurevich,
V.V., and Gurevich, E.V. (2004) Trends Pharmacol
Sci 25 (2).
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Crystal structure of visual arrestin
Hirsch, Schubert, Gurevich, Sigler. Cell 97,
257-269 (1999)
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1.9A crystal structure of arrestin2 (a.k.a.
b-arrestin1)
Han, Gurevich, Vishnivetskiy, Sigler, Schubert.
Structure 9, 869-880 (2001)
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Visual arrestin and arrestin2 almost identical
twins?
Han, Gurevich, Vishnivetskiy, Sigler, Schubert.
Structure 9, 869-880 (2001)
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A major conformational rearrangement of
arrestin is necessary to bring the elements in
both domains implicated in receptor binding into
contact with the cytoplasmic tip of the receptor.
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Critical interactions keep arrestin in its basal
state
Hirsch, Schubert, Gurevich, Sigler. Cell 97,
257-269 (1999) Figure from Gurevich and Gurevich
(2004) Trends Pharmacol Sci 25 (2).
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Key salt bridge between Arg175 and Asp296
functions as a phosphate sensor in the
polar core of arrestin
Vishnivetskiy, Paz, Schubert, Hirsch, Sigler,
Gurevich. J. Biol. Chem. 274, 11451-4 (1999)
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Stabilizing three-element interaction between
b-strand I and a-helix I in the N-domain and
arrestin C-tail is also disrupted by
receptor-attached phosphates.
Vishnivetskiy, Schubert, Climaco, Gurevich,
Velez, Gurevich. J. Biol. Chem. 275, 41049-57
(2000)
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Arrestin binding to the receptor involves the
movement of the two domains relative to each
other and the release of arrestin C-tail.
Figure from Gurevich and Gurevich (2004) Trends
Pharmacol Sci 25 (2).
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• Arrestin promotes receptor internalization
  via coated pits
• Arrestin binds clathrin (Goodman, Krupnick,
  Santini, Gurevich, Penn, Gagnon, Keen, Benovic
  (1996) Nature 383, 447-450)
• 2. Arrestin binds clathrin adapter AP2 (a.k.a.
  adaptin-2) (Laporte, Oakley, Holt, Barak, Caron
  (2000) J. Biol. Chem. 275, 23120-23126)
• 3. Arrestin interacts with NSF (N-ethylmaleimide-s
  ensitive fusion protein) (McDonald, Cote, Lin,
  Premont, Pitcher, Lefkowitz (1999) J. Biol. Chem
  274, 10677-10680)

Figure from Gurevich and Gurevich (2003)
Structure 11, 1037-1042.
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Receptor trafficking down the slippery slope or
there and back again?

• Proteinase-activated receptors (PARs) disposable
  by design.
• (Reviewed in MacFarlane et al. (2001) Pharmacol.
  Rev. 53, 245-282)
• 2. Class A receptors arrestin3gtarrestin2gtgtgtvisu
  al arrestin
• Arrestin dissociates near the plasma
  membrane (in early endosomes?)
• Examples b2-adrenergic receptor, m-opioid
  receptor, D1A dopamine receptor.
• Class B receptors arrestin2arrestin3gtvisual
  arrestin
• They stay in complex with arrestin for a
  long time (in late endosomes and/or perinuclear
  recycling compartment?)
• Examples Angiotensin AT1a, vasopressin V2, TRH
  receptor, substance P receptor.
• (Receptor classification Oakley, Laporte, Holt,
  Caron, Barak (2000) J. Biol.Chem. 275,
  17201-17210)

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The long and the short of receptor recycling
(Innamorati, Gouill, Balamotis, Birnbaumer (2001)
J. Biol.Chem. 276, 13096-13103).
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To recycle or not to recycle, that is the
question. How does the cell decide the fate of
internalized receptor?
?
?
?
?
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Arrestin promotes receptor ubiquitination serving
as an adapter for ubiquitin ligase Mdm2.
Ubi
Ubi
Ubi
Mdm2
Mdm2
Arrestin ubiquitination stabilizes
arrestin-receptor complex. Does receptor
ubiquitination facilitate its down-regulation?
Shenoy, McDonald, Kohout, Lefkowitz (2001)
Science 294, 1307-1313. Shenoy and Lefkowitz
(2003) J. Biol. Chem. 278, 14498-506.
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By targeted mutations in the two hot spots in
arrestin molecule we can make phosphorylation-inde
pendent version of any arrestin For example,
R169E mutation in arrestin2 is analogous to R175E
in visual
Kovoor, Celver, Abdryashitov, Chavkin, Gurevich
(1999) J. Biol. Chem. 274, 6831-6834. Celver,
Vishnivetskiy, Chavkin, Gurevich (2002) J. Biol.
Chem. 277, 9043-9048. Figure from Pan, Gurevich,
and Gurevich (2003) J. Biol. Chem. 278,
11623-11632.
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Phosphorylation-independent arrestins prevent
receptor down-regulation.
Does the length of receptor tenure in endosomes
determine its fate?
Pan, Gurevich, and Gurevich (2003) J. Biol. Chem.
278, 11623-11632.
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Signaling switch 1 a simple feedback loop
b2AR
Phospho-b2AR
Gi
Gs
Inhibition
Stimulation
Adenylyl cyclase
cAMP
PKA
Daaka, Luttrell, Lefkowitz (1997) Nature 390,
88-91.
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Signaling switch 2 mopping up
GRK
b2AR
b2AR
b2AR
Arrestin
P
P
P
P
P
P
Arrestin
PDE4D3
Gs
PDE4D3
Hydrolysis
Adenylyl cyclase
cAMP
Synthesis
(Perry, Baillie, Kohout, McPhee, Magiera, Ang,
Miller, McLean, Conti, Houslay, Lefkowitz (2002)
Science 298, 834-836)
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Signaling switch 3 MAP kinases break loose
Non-visual arrestins interaction partners
Protein Site in
arrestins Edocytic proteins Clathrin
C-terminus AP2 C-terminus NSF
? Kinases Src N-terminus/Central
Region JNK3 Central
region ASK1 N-terminus ERK1/2 ? Raf
?
Reviewed in MillerLefkowitz (2001) Curr. Opin.
Cell Biol. 13, 139-145.
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Another feedback loop ERK shoots back.
GRK
PP
b2AR
b2AR
b2AR
b2AR
P
Arrestin2
Arrestin2
P
P
P
P
P
P
P
P
P
P
Arrestin2
Gs
b2AR
Arrestin2
P
P
P
Clathrin
Signaling
AP2
Src
b2AR
Clathrin
P
?
Arrestin2
P
P
P
AP2
?
ERK
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Summary (incomplete)
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Glossary
1. Desensitization - loss of responsiveness to a
persistent stimulus. 1A. Heterologous
desensitization - desensitization of a receptor
that was not activated. It is usually mediated by
second messenger-activated kinases. Examples
b2-adrenergic receptor. phosphorylation by PKA in
response to any increase in cAMP m2 muscarinic
cholinergic receptor phosphorylation by PKC in
response in elevated intracellular Ca2. This
phosphorylation does not promote arrestin binding
(wrong place? too few phosphates?). 1B.
Homologous desensitization - desensitization only
of the receptor that is being stimulated.
Mediated by selective GRK phosphorylation of
activated receptor followed by arrestin binding
to phosphorylated activated receptor.
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2. Internalization (endocytosis) - removal of
the receptor away from plasma membrane (to
endosomes). Possible routes 2A. Via coated
pits (dynamin-dependent, arrestin-dependent).
Best studied example b2AR. 2B. Via caveoli
(dynamin-dependent, arrestin-independent).
Example m2 mAChR. 2C. Via an alternative
route (dynamin-independent) (not really
well-defined or studied). 3. Down-regulation -
the loss of the total number of receptor (due to
its degradation in lysosomes). Examples
virtually any GPCR. Proteinase-activated
receptors (PARs) are always degraded upon
internalization.
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4. Resensitization - the restoration of cell
sensitivity after desensitization. In case of
b2AR resensitization requires receptor
internalization, arrestin dissociation, receptor
dephosphorylation and recycling back to the
plasma membrane. Some other GPCRs can be returned
to their original state (resensitized) without
internalization. 5. Recycling - the return of the
internalized receptor to the plasma membrane.
Varying proportion of internalized receptor can
be recycled or targeted to lysosomes and
degraded. The cell apparently has mechanisms to
decide what to do with it (that we dont know
much about).