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Departmental Student Presentation
Steven A. Moore Jr.
Identification of Amino Acid Motifs in the Human Angiotensin II Type-2
Receptor Involved in G-protein Activation
Honors Biology Presentation
2002
Weyhenmeyer Laboratory
Dept. of Cell and Structural Biology
University of Illinois
512 Medical Science Building
(217) 333-8075
[email protected]
Steve’s Thesis
Problem:
What are the primary, secondary, and tertiary structures in the human AT2
receptor that are necessary for G-protein coupling and activation?
Steve’s Thesis
Problem:
What are the primary, secondary, and tertiary structures in the human AT2 receptor that
are necessary for G-protein coupling and activation?
Background Information:
Concept Map
Angiotension II Peptide
Hormone
Angiotensin II Type-1
Membrane Receptor
Angiotensin II Type-2
Membrane Receptor
G-protein Coupled Membrane Receptor
Superfamily
Introduction to Angiotensin II Hormone
(Ang II)
• Angiotensin II octapeptide is a naturally occurring hormone:
• Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
• Sar1Ile8-Angiotensin II is a synthetic version of Angiotension II :
• Sar-Arg-Val-Tyr-Ile-His-Pro-Ile
• Best known for blood pressure regulation, body fluid homeostasis, and
electrolyte balance (AT1R mediated).
• Also found to influence cell growth, differentiation, and death.
The G-Protein Coupled Receptor(GPCR) Superfamily
 All G-protein receptors are homologous, this has become clear from DNA
sequencing experiments.
 The amino acid sequences of a large number of these receptors reveal a common
structure consisting of a single polypeptide chain that threads back and forth
across the lipid bilayer seven times.
 Common structural motifs found in all GPCRs include:
1.) Seven a-helical hydrophobic transmembrane regions
2.) Three extracellular loops and tail
3.)Three intracellular loops and tail
4.)A variant of the DRY motif, a highly conserved motif thought to be involved in
G-protein coupling.
NH2
E1
I
II
III
IV
D D
R
Y
I1
E3
E2
VI
V
I
I2
VII
K
K
L
K
K
OUT
I3
IN
Q
K
N
HOOC
Fig. 2) Diagram of the human AT2 receptor including the seven transmembrane domains along with intra and extra-cellular loops and tails.
Proposed amino acids to be mutagenized are highlighted and their single letter designations are adjacent.
Properties of Angiotensin II Receptor Subtypes
AT1
AT2
Agonist:
Ang II
Ang II
Localization:
liver, cortex, HTSM,
lung, heart, spleen,
uterus, adrenal gland,
v.s.m, kidney
adrenal gland, uterus,
kidney, heart, pancreas,
cerebellum, HTSM,
cerebral vessels
Peripheral function:
vasoconstriction,
adrenal aldosterone
and catecholamine
release, enhanced NE
release from sym. ganglia
growth, development,
wound healing
AII
Step 1) Angiotensin II binding to high affinity site,
and subsequent AT2 receptor activation.
AII
GGDP
Step 2) Newly assembled G-protein
AII
Step 4) hormone
is released from
low affinity binding
site after G-protein
binds to active
receptor.
binds active AT2 receptor.
AII
GGDP
Step 3) GDP exchanged for GTP
GTP
GDP
Inactive receptor
Step 5) Active G-protein activates next enzyme in pathway.
GGDP
Fig 1.) G-protein activity diagram.
?
Introduction to Angiotensin II type-2 Receptor (AT2R)
 Member of the G-protein Coupled Receptor(GPCR) superfamily.
 Agonist: Angiotensin II Hormone
Partial Agonist: Sar1Ile8-Angiotensin II
Specific Antagonist: PD123,319 ( blocks the receptor for Angiotension II)
 Major structural motifs include:
1.) 7 hydrophobic transmembrane regions.
2.) Hormone binding regions comprised of 3 extracellular loops and tail.
3.) G-protein binding regions comprised of 3 intracellular loops, including
the DRY motif, and cytoplasmic tail.
NH2
E1
I
II
III
IV
D D
R
Y
I1
E3
E2
VI
V
I
I2
VII
K
K
L
K
K
OUT
I3
IN
Q
K
N
HOOC
Fig. 2) Diagram of the human AT2 receptor including the seven transmembrane domains along with intra and extra-cellular loops and tails.
Proposed amino acids to be mutagenized are highlighted and their single letter designations are adjacent.
Introduction to Angiotensin II type-2 Receptor (AT2R)
 Member of the G-protein Coupled Receptor(GPCR) superfamily.
 Agonist: Angiotensin II Hormone
Partial Agonist: Sar1Ile8-Angiotensin II
Specific Antagonist: PD123,319 ( blocks the receptor for Angiotension II)
 Major structural motifs include:
1.) 7 hydrophobic transmembrane regions.
2.) Hormone binding regions comprised of 3 extracellular loops and tail.
3.) G-protein binding regions comprised of 3 intracellular loops, including
the DRY motif, and cytoplasmic tail.
 AT2 receptor has been shown to be involved in:
Inhibition of cell proliferation (Tsuzuki et al., 1996)
Development (Mukoyama et al., 1993)
Apoptosis (Yamada et al., 1996)
Neuronal differentiation (Schelman et al., 1997, Bedecs et al., 1997)
Fluorescent Stained Neuron
Steve’s Thesis
Problem:
What are the primary, secondary, and tertiary structures in the human AT2
receptor that are necessary for G-protein coupling and activation?
Hypothesis:
1.) If the DRY motif (Asparagine, Arginine, Tyrosine), found in the human AT2
receptor, is a key player in G-protein activation, then mutation of this motif should
significantly effect the receptors ability to activate G-proteins.
2.) If a specific sequence of the AT2 receptor is key to binding G-proteins, then we
can identify regions by using synthetic proteins to compete with the active receptor
for the G-protein.
Experiments to test Hypotheses:
 1a). Determine whether mutations in the DRY motif of the AT2 receptor effect
AngII hormone binding affinity or receptor expression levels?
• 1b.) Determine whether mutations in the DRY motif effects the receptor’s
ability to bind and activate G-proteins.
 2.) Map the G-protein binding domains of the AT2 receptor using synthetic
peptides selected from the receptor sequence.
1a.) Mutation, transfection, and characterization of wild type
and DRY motif mutant receptors.
Site-directed mutagenesis of human AT2 receptor. (PCR based method)
Insert wild type and mutant receptor DNA into Chinese Hamster Ovary cells.
(Lipid based transfection protocol)
Radioactively tagged ligand binding analysis of wild type and mutant receptors.
Scatchard analysis will be used to calculate binding affinity and expression levels
for each receptor.
Fig.5) Scatchard Analysis: Sar-Ile Binding Affinity for Wild
Type and Mutant AT 2 Receptors Transiently Expressed
in CHO-K1 cells.
0.0125
Wild Type
D-A
0.01
R-A
Y-A
DRY
B/F Specific
0.0075
0.005
0.0025
Bound
2.
50
00
E11
1
2.
00
00
E1
1.
50
00
E1
1
1.
00
00
E11
2
5.
00
00
E1
0.
00
00
E+
00
0
Fig.6) Binding Affinity of Wild Type and Mutant
Receptors for AngII and Sar-Ile
10
SarIle
1
D
R
Y
-A
A
A
Receptor
Y
-A
R
-A
D
-A
t
0.1
W
kD (nm)
Ang II
Fig.7) Bmax of Wild Type and Mutant Receptors with
AngII and Sar-Ile
500
300
SarIle
Ang II
200
100
D
R
Y
-A
A
A
Receptor
Y
-A
R
-A
D
-A
t
0
W
Bmax (fm/mg)
400
CHO
Wt
D141-A
R142-A
Y143-A
DRY-AAA
Western blot of CHO cells transfected with wild type and mutant AT2 receptors:
Lane 1.): non-transfected CHO-K1 cells, 2.) Wild type human AT2 receptor,
3.) D141-A mutant receptor, 4.) R142-A mutant receptor, 5.) Y143-A mutant receptor,
6.) DRY-AAA mutant receptor
1b.) Effects of GTPgS on AT2 receptor's Ang II binding affinity.
  GTPgS will decrease a G-protein coupled receptor's affinity for its ligand by
maintaining the receptor in an active state.
  Wild type receptor is expected to have the largest decrease in Ang II affinity
with the addition of GTPgS. Wild type receptor % decrease of Ang II affinity
with GTPgS will be the positive control.
AII
Step 1) Angiotensin II binding to high affinity site,
and subsequent AT2 receptor activation.
AII
GGDP
Step 2) Newly assembled G-protein
AII
Step 4) hormone
is released from
low affinity binding
site after G-protein
binds to active
receptor.
binds active AT2 receptor.
AII
GGDP
Step 3) GDP exchanged for GTP
GTP
GDP
Inactive receptor
Step 5) Active G-protein activates next enzyme in pathway.
GGDP
Fig 1.) G-protein activity diagram.
?
Effects of GTP gS on AngII Binding Affinity
125
75
% Max S hift
50
25
D
R
Y
-A
A
A
Receptor
Y
-A
R
-A
D
-A
t
0
W
% Max Shift
100
2.) Use of synthetic peptides to identify key regions of
G-protein coupling.
  If a specific sequence of the AT2 receptor is key to binding G-proteins, then we
• can identify regions by using synthetic proteins to compete with the active receptor
for the G-protein.
•
  This protocol has also been used to map G-protein coupling regions in the badrenergic (Strader et al., 1987; O'Dowd et al., 1988; Munch et al., 1991), a1adrenergic (Cotecchia et al., 1990), rhodopsin (Konig et al., 1989), and the m2
muscarinic receptors (McClue et al., 1994).
  Advantages and Disadvantages vs. Site-directed Mutagenesis
Conclusions
1a.)
 Wild type receptors can be mutated via a PCR based site-directed mutagenesis
protocol. We have also demonstrated that high transient expression levels can be
obtained using a lipid based transfection protocol.
 Even more importantly, we have been able to determine that the mutations in the
DRY motif did have a significant effect on the receptor's Ang II affinity, and in some
cases expression levels.
1b.)
 We have shown that GTPgS decreases Ang II affinity in all of the receptors tested.
This demonstrates that all receptors are coupling with G-proteins. As expected, the wild
type receptor had the largest decrease of affinity, signifying it's strong affinity for Gproteins.
2.)
• We have demonstrated that we can map key G-protein coupling regions
using synthetic proteins to compete with active receptor.
Thanks:
James Weyhenmeyer, Ph.D.
Anjali Patel
Nancy Huang
Bridget Lavin
Jungsik Yo
Paul Ferguson
Tom Grammatopoulos
Rob Andres
Steve’s Lab
Steve’s Poster Presentation
Incubating the CHO
Examining Cells
Feeding the CHO
Frozen CHO
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