Signaling and Regulation of Protease-Activated Receptors 1 and 2 (PAR1 and PAR2) Público

McCoy, Kelly Lynn (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/6969z0995?locale=es
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Abstract


Signaling and Regulation of Protease-Activated Receptors 1 and 2 (PAR1 and
PAR2)

By Kelly L. McCoy
Unique aspects of PAR1 and PAR2 include their distinctive mechanism of activation,
their multiplicity of G protein coupling, their differential signal regulation, and their
contribution to remarkably diverse cellular processes. Recent evidence suggests that these
closely related receptors regulate different physiological outputs in the same cell, though
little is known about their comparative signaling pathways. Here we report that PAR1 and
PAR2 couple to overlapping and distinct sets of G proteins to regulate receptor-specific
signaling pathways involved in cell migration. We also investigated potential regulatory
mechanisms in place to fine-tune PAR1 and PAR2 signaling. Their signaling must be
tightly controlled since they are irreversibly activated and stimulate multiple G protein-
linked pathways. My studies demonstrate that selective, cell type-specific G protein
coupling to PAR1 and/or PAR2 may provide one such level of regulation. An additional
level of PAR regulation may come from regulators of G protein signaling (RGS) proteins,
which act as GTPase-activating proteins and thereby inhibit G protein signaling. Little is
known about RGS regulation of PAR signaling but my data using purified proteins and
intact cells suggest that RGS proteins have both distinct and overlapping effects on PAR1
and PAR2, including a capacity to modulate functional readouts of PAR signaling.
Together, these studies demonstrate that PAR1 and PAR2 functionally couple to
overlapping and distinct G protein-linked functional pathways, and these signaling events
are regulated, at least in part, by specific RGS proteins in receptor- and G protein-
dependent manners.


Signaling and Regulation of Protease-Activated Receptors 1
and 2 (PAR1 and PAR2)


By
Kelly L. McCoy
B.S., Vanderbilt University, 2004
Advisor: John R. Hepler, Ph.D.
A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies
of Emory University in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in Molecular and Systems Pharmacology
Graduate Division of Biological and Biomedical Sciences
2010

Table of Contents


Table of Contents

CHAPTER 1: Introduction

.............................................................................................. 1
1.1. Cardiovascular Disease and Stroke ...................................................................... 2
1.1.1. Overview............................................................................................................ 2
1.1.2. Stroke prevention in individuals living with cardiovascular disease................ 2
1.2. Protease-Activated Receptors ............................................................................... 4
1.2.1. Overview............................................................................................................ 4
1.2.2 Conventional GPCR Activation and Signaling .................................................. 9
1.2.3. PAR Activation and Signaling......................................................................... 10
1.2.4. PARs in Physiological Processes.................................................................... 16
1.3. RGS Protein Overview......................................................................................... 24
1.3.1. RGS protein structure determines function..................................................... 25
1.3.2. RGS protein interactions with GPCRs ............................................................ 26
1.4. Rationale and Objectives for this Dissertation .................................................. 37
CHAPTER 2: PAR1 and PAR2 couple to overlapping and distinct sets of G proteins
and linked signaling pathways to differentially regulate cell physiology
................... 40
2.1. Introduction .......................................................................................................... 41
2.2. Experimental Procedures .................................................................................... 43
2.2.1. Materials ......................................................................................................... 43
2.2.2. Methods ........................................................................................................... 44
2.3. Results ................................................................................................................... 50
2.3.1. PAR1 and PAR2 link to multiple G protein-regulated pathways.................... 50
2.3.2. PAR1 and PAR2 form stable complexes with both overlapping and distinct
sets of G proteins
....................................................................................................... 56
2.3.3. PAR1 and PAR2 form stable complexes with G protein heterotrimers .......... 60
2.3.4. PAR1 selectively couples to Gi/o signaling pathways...................................... 62
2.3.5. PAR1 and PAR2 both utilize Gq/11 and G12/13 to activate PLC and Rho,
respectively
................................................................................................................ 65
2.3.6. PAR-stimulated cAMP, PLC and RhoA signaling in Neu7 cells..................... 66
2.3.7. PAR1 and PAR2 utilize overlapping and distinct G protein pathways to
stimulate ERK1/2 phosphorylation in Neu7
.............................................................. 72
2.3.8. PAR1, but not PAR2, influences Neu7 cell migration via a PTX-sensitive Gi/o
pathway
..................................................................................................................... 74
2.4. Discussion.............................................................................................................. 77
2.4.1. PAR1 and PAR2 both couple to multiple overlapping sets of G proteins....... 78
2.4.2. PAR1, but not PAR2, couples to Go and also to Gi family members............... 79
2.4.3. PAR1 and PAR2 form complexes with G proteins that are stable in the
presence of agonist and nucleotide
........................................................................... 80
2.4.4. Gi/o signaling mediates PAR1 but not PAR2 contributions to ERK1/2 signaling
and migration in Neu7 astrocytes
............................................................................. 82
Chapter 3: Point mutations in the second intracellular loop of PAR1 selectively
disrupt receptor coupling to G

.......................................... 84
q/11 but not to Gi/o or G12/13
3.1. Introduction .......................................................................................................... 85
3.2. Experimental Procedures .................................................................................... 88
3.2.1. Materials ......................................................................................................... 88


3.2.2. Methods

........................................................................................................... 89
3.3. Results ................................................................................................................... 92
3.3.1. Five amino acid residues in the PAR1-i2 loop are important for the receptor's
capacity to activate inositol phosphate signaling
..................................................... 93
3.3.2. PAR1 mutants that disrupt Gq/11 coupling do not affect PAR1 coupling to Gi/o-
or G

..................................................................................................................... 95
12/13
3.3.3. Some PAR1 mutant receptors have reduced capacities to bind to G11, but not
to G

............................................................................................................ 100
o, or G12
3.3.4. PAR1 i2 loop mutants have differential capacities to stimulate calcium
signaling in astrocytes from PAR1-/- mice
............................................................. 102
3.4. Discussion............................................................................................................ 106
3.4.1. Five discrete point mutations within the PAR1 i2 loop reduce the capacity of
the receptor to stimulate Gq/11/PLC-mediated inositol lipid signaling
................... 108
3.4.2. The five PAR1 i2 loop point mutations that disrupt InsP signaling have no
effect on G12/13-mediated RhoA activation or Gi/o-stimulated ERK1/2
phosphorylation
....................................................................................................... 109
3.4.3. Of the five point mutations that reduce PAR1/Gq/11 functional coupling, only
two prevent PAR1 and G11 from forming a complex
............................................... 110
3.4.4. A single point mutation in the PAR1 i2 loop disrupts downstream calcium
mobilization in astrocytes from PAR1-/- mice
........................................................ 110
Chapter 4: RGS protein regulation of PAR1 and PAR2 is RGS-, receptor-, and G
protein-dependent
......................................................................................................... 113
4.1 Introduction ......................................................................................................... 114
4.2. Experimental Procedures .................................................................................. 116
4.2.1. Materials ....................................................................................................... 116
4.2.2. Methods ......................................................................................................... 117
4.3 Results .................................................................................................................. 120
4.3.1. RGS2 and RGS4 differentially interact with PAR1 and PAR2...................... 121
4.3.2. Cytoplasmic i2 and i3 loops of PAR1 but not PAR2 are involved in
interactions with RGS proteins
............................................................................... 125
4.3.3. RGS2 and RGS4 but not RGS1 block PAR-activated calcium-activated
chloride currents in oocytes
.................................................................................... 128
4.3.4. RGS2 and RGS4 differentially regulate PAR1- and PAR2-stimulated ERK1/2
phosphorylation
....................................................................................................... 130
4.3.5. RGS4 and RGS16 regulate PAR2- but not PAR1-stimulated RhoA activation
................................................................................................................................. 132
4.4 Discussion............................................................................................................. 134
4.4.1. PAR1 and PAR2 interact with overlapping and distinct sets of RGS proteins in
receptor- and G protein-dependent manners
.......................................................... 134
4.4.2. RGS proteins regulate PAR1 but not PAR2 by interacting with its i2 and i3
loops
........................................................................................................................ 137
4.4.3. PAR1 and PAR2 signaling is differentially regulated by RGS proteins at the
level of the receptor and the level of the G protein-linked signaling pathway
....... 140
CHAPTER 5: Discussion .............................................................................................. 142
5.1. PAR1 and PAR2 couple to overlapping and distinct sets of G proteins ....... 143
5.1.1. PAR1 but not PAR2 interacts with Gi/o family members ............................... 143


5.1.2. PAR1 but not PAR2 interacts with Gi/o-linked signaling pathways

.............. 144
5.2. Arg205 amino acid residue in the PAR1 i2 loop dictates receptor binding to
G

............................................................................................. 148
11 but not to Go or G12
5.3. RGS regulation of PAR1 and PAR2 is receptor- and G protein- dependent 150
5.3.1. PAR1 and PAR2 interact with overlapping and distinct sets of RGS proteins
................................................................................................................................. 151
5.3.2. PAR1 and PAR2 signaling is differentially regulated by RGS proteins at the
level of the receptor and the level of the G protein-linked signaling pathway
....... 152
5.4. Overall Conclusions and Implications.............................................................. 154
5.5. Future Directions................................................................................................ 157




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