Protein-Protein Interaction Motifs in GPCR Presented by: Harpreet K Dhiman Research Associate, Dept. of Pharmacology, Univ. of Pittsburgh

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Protein-Protein Interaction Motifs in
GPCRPresented byHarpreet K DhimanResearch
Associate,Dept. of Pharmacology, Univ. of
Pittsburgh
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Motifs in GPCR

• Introduction
• to
• Protein-Protein Interactions
• in
• GPCR

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Motifs in GPCR
GPCR Interactions Occur in Cytoplasmic Loop
3rd
15th
11th
7th
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Motifs in GPCR

• Receptors Divided into Various Classes based on
  Sequence Homology
• GPCR classes
• Class A Rhodopsin like
• Class B Secretin like
• Class C Metabotropic glutamate / pheromone
• Class D Fungal pheromone
• Class E cAMP receptors (Dictyostelium)
• Frizzled/Smoothened family
• Putative Families
• Ocular albinism proteins
• Drosophila odorant receptors
• Plant Mlo receptors
• Nematode chemoreceptors
• Vomeronasal receptors (V1R V3R)
• Orphans
• Putative / unclassified GPCRs

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Motifs in GPCR

• Goal
• Identify Motifs to Find Potential
• Protein-Protein Interactions in
• GPCR s

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Motifs in GPCR

• Approach
• Step 1 List proteins that interact with GPCRs
  based on experimental proteomic approach
  (affinity chromatography and mass spectrometry)
• Step 2 Identify domains in these proteins using
  Swiss-prot (web based tool)
• Step 3 Select domains that have known
  protein-protein interaction modules
• Step 4 Identify general Motifs for PDZ and SH3
  domains from research articles

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Motifs in GPCR

• Step 1 List Proteins that Interact with GPCRs
  based on experimental proteomic approach
  (affinity chromatography and mass spectrometry)
• VeLi3
• Post-synaptic density protein 95 (PSD95)
• Dlgh3 protein (MPP3)
• Calmodulin
• F-actin capping protein ß subunit (CAPZ ß)
• F-actin capping protein a-2 subunit (CAPZ a-2)
• PKC ?-interacting protein PICOT
• Actin, cytoplasmic 1 (ß-actin)
• 2810409H07Rik protein
• Dynamin 1
• SPectrin a II chain (a-fodrin)

Source Synaptic multiprotein complexes
associated with 5-HT2C receptors a proteomic
approach. Becamel et al, The EMBO Journal, Vol
21, No. 10 pp 2332-2342, 2002
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Motifs in GPCR

• Step 2 Identify Domains in these proteins using
  Swiss-prot (web-based tool)

Protein Function
Veli3 One of vertebrate homologues of LIN-7 essential for vulval development, enriched in brain.
PSD95 Clustering of its binding partners at cell surface, targeting of its PDZ ligands
MPP3 Important for synaptic localization of its binding partners (invivo)
Calmodulin Calcium ion binding
CAPZ- ß Capping proteins that binds as a dimer to the barbed end of actin filaments and inhibits the growth of actin microfilaments
CAPZ a-2 Capping proteins that binds as a dimer to the barbed end of actin filaments and inhibits the growth of actin microfilaments
PICOT Interacts with PKC-related kinases plays role in regulating function of thioredoxin system
ß-actin Involved in the formation of filaments that are major components of the cytoskeleton
2810409H07Rik GTP-binding protein important in development and normal cell metabolism
Dynamin I Microtubule-associated, force-producing protein involved in production of microtubule bundles
a-fodrin Serves as a docking surface for cytoskeletal and signal transduction proteins
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Motifs in GPCR

• Step 3 Select Domains with known Protein-Protein
  Interaction Modules

Protein Domain Domain Domain Domain Domain Domain Domain Domain Domain Domain Domain Domain Domain Domain Domain Domain
PDZ SH3 L27 GuanylateKinase PTS EF Hand F_ Actin capA F_ actin capB Thio_ redoxin Actin GTP1OBG Dynamin PH GED G_ptotein _recep_ f1_1 Spectrin
Veli3 - - - - - - - - - - - - - -
PSD95 - - - - - - - - - - - -
MPP3 - - - - - - - - - - - -
Calmodulin - - - - - - - - - - - - - - -
CAPZ- ß - - - - - - - - - - - - - - -
CAPZ a-2 - - - - - - - - - - - - - - -
PICOT - - - - - - - - - - - - - - -
ß-actin - - - - - - - - - - - - - - -
2810409H07Rik - - - - - - - - - - - - - - -
Dynamin I - - - - - - - - - - - - -
a-fodrin - - - - - - - - - - - -
Green Aid in protein-protein interaction Red
Dont aid in protein-protein interaction
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Motifs in GPCR

• SH3 Domain

Structure and Binding The basic fold of SH3
domains contains five anti-parallel ß-strands
packed to form two perpendicular ß-sheets.
Src-homology 3 (SH3) domains generally bind to
Pro-rich peptides that form a left-handed polyPro
type II helix, with the minimal consensus
Pro-X-X-Pro. Each of these aliphatic-Pro pairs
binds to a hydrophobic pocket on the SH3 domain.
From this, two classes of SH3 domains have been
defined (Class I and Class 2) which recognize
RKXXPXXP and PXXPXR motifs respecitvely. The
ligand can, in principle, bind in either
orientation. Directionality is conferred by the
interaction of the Arg or Lys residue with the
charged outer face of the SH3 domain while the
tandem prolines bind within two hydrophobic
pockets of the SH3 domain.
The figure shows a Sem5 C-terminal SH3 domain
complexed to the mSos-derived sequence PPPVPPRRR.
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Motifs in GPCR

• PDZ Domain

Structure and Binding PDZ domains contain 80-90
residues that fold into a structure with a
ß-sandwich of 5-6 ß-strands and two a-helices.
The peptide ligand binds in a hydrophobic cleft
composed of a ß-strand (ßB), an a-helix and a
loop that binds the peptide carboxylate group.
The peptide binds in an anti-parallel fashion to
the ßB strand, with the C-terminal residue
occupying a hydrophobic pocket. PDZ heterodimers
form a linear head-to-tail arrangement that
involves recognition of an internal on one of the
partner proteins.
The figure shows the 3rd PDZ domain of PSD-95
bound to a TKNYKQTSV peptide.
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Motifs in GPCR
Binding to Internal Motifs Internal motifs can
be recognized if they are presented within a
specific tertiary structure context that
conformationally mimics a chain terminus.
Fig. The same PDZ domain can recognize two
structurally distinct ligands. The a1-syntrophin
PDZ domain is shown as a gray solvent-accessible
surface. (A) The ligand is canonical peptide
NH2-VKESLV-COOH, shown as a red tube (Schultz et
al., 1998). (B) The ligand is the nNOS PDZ domain
with its distinctive b-finger motif (indicated),
shown as a red ribbon diagram. The internal
peptide motif that mimics a C-terminal peptide is
indicated (Hillier et al., 1999). Source
http//www.ucsf.edu/limlab/papers/bzh_2001b.pdf
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Motifs in GPCR

• Internal Motif Recognition
• e.g. nNOS-syntrophin complex
• Interaction mediated by contacts between PDZ
  domain of syntrophin and a ß-finger in the PDZ
  flanking region of nNOS
• First strand of nNOS ß-finger (sequence - ETTF- )
  mimics a canonical C-terminal peptide in its
  sequence specific interaction with the
  peptide-binding grove of syntrophins PDZ
• C-terminal free carboxylate group is replaced by
  sharp ß-turn at the tip of ß-finger

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Motifs in GPCR
Internal Motif Recognition (ß-finger
required) Structure of PDZ and PDZ-like domains
A Ribbon diagram of PDZ3 of PSD-95 complexed
with a C-terminal peptide from CRIPT. The
structure demonstrates the six ß-strands
(turquoise) and two a-helices (red) with the
peptide (yellow) binding as a ß-strand between
the a B helix and ßB strand.
B Structure of a1-syntrophin PDZ domain
complexed to nNOS (green). Note the overall
similarity in PDZ structure with replacement of
the C-terminal peptide ligand by a ß-finger.
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Motifs in GPCR

• Step 4 Identify general Motifs for PDZ and SH3
  domains from Research Articles
• General Motifs Identified for PDZ Domain

Domain Regular Expression
PDZ xSTxVLend
PDZ xVLIFYWMxVLIFYWMend
PDZ xDExVLIFYWMend
PDZ FYSTxFVAend
PDZ ESTxVLIFYWMend
PDZ EVLIFYWMxVLIFYWMend
PDZ DxxC
PDZ-I ESTxVIend
PDZ-II DxV
PDZ-III YFWYFWVLIFYWMend
PDZ xDxV
PDZ DESTxLVMI
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Motifs in GPCR

• Step 4 contd.
• General Motifs Identified for SH3 Domain

Domain Regular Expression
SH3 RxVLIFYWMPxxP
SH3 PxxPxR
SH3 RKxxPxxP
SH3 PxxPxRK
SH3 PxxDY
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Motifs in GPCR

• RESULTS
• PDZ domain recognizing motifs were found to be
  present in all classes of GPCRs (Class through
  Class F)
• SH3 domain recognizing motifs were found in all
  classes of GPCRs except Class E.

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Motifs in GPCR
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Motifs in GPCR
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Motifs in GPCR

• Some Examples..
• Receptors with PDZ domains in Class A

b1ar Beta Adrenoceptors type 1
5h2a Serotonin Vertebrate type 2
clt2 Cysteinyl leukotriene
Dbdr Dopamine Vertebrate type 1
Oxyr Oxytocin
5h1a Serotonin Vertebrate type 1
5h2b Serotonin Vertebrate type 2
A1aa Alpha Adrenoceptors type 1
A1ad Alpha Adrenoceptors type 1
b1ar Beta Adrenoceptors type 1
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Motifs in GPCR

• Some Examples..
• Receptors with SH3 domains in Class A

Ckra C-C Chemokine type 10
5h6 Serotonin Vertebrate type 6
A2aa Alpha Adrenoceptors type 2
B3ar Beta Adrenoceptors type 3
D2dr Dopamine Vertebrate type 2
Ox2r Orexin
Ur2r Urotensin II
5h1a Serotonin Vertebrate type 1
5h6 Serotonin Vertebrate type 6
5ht Serotonin Insect type 1
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Motifs in GPCR

• Some Examples..
• Receptors with PDZ domains in Class B

bai1 Brain-specific angiogenesis inhibitor (BAI)
Bai3 Brain-specific angiogenesis inhibitor (BAI)
Stan Cadherin EGF LAG (CELSR)
Bai2 Brain-specific angiogenesis inhibitor (BAI)
Cgrr Calcitonin
Mth1 Methuselah-like proteins (MTH)
Clr1 Cadherin EGF LAG (CELSR)
Clr3 Cadherin EGF LAG (CELSR)
Glr Glucagon
Calr Calcitonin
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Motifs in GPCR

• Some Examples..
• Receptors with SH3 domains in Class B

Clr3 Cadherin EGF LAG (CELSR)
Mth4 Methuselah-like proteins (MTH)
clr1 Cadherin EGF LAG (CELSR)
Clr2 Cadherin EGF LAG (CELSR)
Crfl Corticotropin releasing factor
Crf2 Corticotropin releasing factor
Scrc Secretin
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Motifs in GPCR

• Some Examples..
• Receptors with PDZ domains in Class C

Mgr1 Metabotropic glutamate group I
Mgr5 Metabotropic glutamate group I
mgr3 Metabotropic glutamate group II
Gbr2 GABA-B subtype 2
Mgr7 Metabotropic glutamate group III
Casr Extracellular calcium-sensing
Mgr2 Metabotropic glutamate group II
Mgr4 Metabotropic glutamate group III
Mgr6 Metabotropic glutamate group III
Mgr8 Metabotropic glutamate group III
Mgr Metabotropic glutamate group II
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Motifs in GPCR

• Some Examples..
• Receptors with SH3 domains in Class C

Casr Extracellular calcium-sensing
Mgr1 Metabotropic glutamate group I
mgr5 Metabotropic glutamate group I
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Motifs in GPCR

• Some Examples..
• Receptors with PDZ domains in Class D

Ste3 Fungal pheromone STE3-like
Bbr1 Fungal pheromone STE3-like
Map3 Fungal pheromone STE3-like
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Motifs in GPCR

• Some Examples..
• Receptors with SH3 domains in Class D

Bbr1 Fungal pheromone STE3-like
Ste3 Fungal pheromone STE3-like
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Motifs in GPCR

• Some Examples..
• Receptors with PDZ domains in Class E

Car3 Class E cAMP receptors (Dictyostelium)
Car1 Class E cAMP receptors (Dictyostelium)
Car2 Class E cAMP receptors (Dictyostelium)
Car4 Class E cAMP receptors (Dictyostelium)
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Motifs in GPCR

• Some Examples..
• Receptors with PDZ domains in Class F

Fzd1 frizzled Group A (Fz 12457-9)
Fzd2 frizzled Group A (Fz 12457-9)
Fzd4 frizzled Group A (Fz 12457-9)
Fzd7 frizzled Group A (Fz 12457-9)
Frz4 frizzled Group C (other)
Fz0a frizzled Group A (Fz 12457-9)
Fz0b frizzled Group A (Fz 12457-9)
Fz10 frizzled Group A (Fz 12457-9)
Fzd6 frizzled Group B (Fz 3 6)
Smo Smoothened
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Motifs in GPCR

• Some Examples..
• Receptors with SH3 domains in Class F

Fzd5 frizzled Group A (Fz 12457-9)
Frz2 frizzled Group A (Fz 12457-9)
Frz4 frizzled Group C (other)
Fz0a frizzled Group A (Fz 12457-9)
Fz0b frizzled Group A (Fz 12457-9)
Fz10 frizzled Group A (Fz 12457-9)
Smo Smoothened
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Motifs in GPCR

• Questions
• ?

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Motifs in GPCR

• Acknowledgements
• Judith Klein Seetharaman
• Betty Cheng