What Are They Looking for? A guide to the Graduate
School Admission Process
Joyce Blair Easter
In Chemistry. 12(2): 23-24, 2002.
Abstract:
Heterotrimeric guanine nucleotide-binding
regulatory proteins (G-proteins) are vital components of numerous
signal transduction pathways, including sensory and hormonal
response systems. G-proteins transduce signals from heptahelical
transmembrane receptors to downstream effectors. The localization
of a G-protein to the plasma membrane, as well as its interaction
with the appropriate receptor and effector, are essential for its
function. In addition, the association of a G-protein's subunits
to form its trimer is required for interaction with its receptor.
The G-protein gamma subunits are subject to a set of
carboxyl-terminal processing events that include prenylation of a cysteine, proteolysis, and
methylation. Recent advances which
elucidate the contributions that the post-translational
modifications of the gamma subunit have on the assembly, membrane
association, and function of the G-protein trimer reveal that
these modifications are required for important protein-protein,
in addition to membrane-protein, interactions.
SAACS Community Service with a Competitive Edge
J.B. Higgins
The Chemical Educator. 3(2): 1-6, 1998.
Introduction:
The student clubs of all the departments housed in the
Physical Sciences Building at Eastern Illinois University united in November of
1996 for a worthy, philanthropic cause and some friendly competition. The
Psychology, Physics, Geosciences, and Chemistry Clubs held a coin war to raise
money for the Charleston Food Pantry. Each club earned points from the
money placed in their department's container. Pennies placed in a
container counted as positive points whereas all other coins and bills counted
as negative points. Point values equaled the face value of the
money. During the two-day event, students, staff, and faculty showed
tremendous spirit and support for their departments. A total of $122.81
was collected, including over $60 in pennies alone. Every cent of the
money collected went to the food pantry to help feed needy families during the
holiday season.
The Role of Prenylation in G-Protein
Assembly and Function (review)
J.B. Higgins and P.J. Casey
Cellular Signalling. 8(6): 433-437, 1996.
Abstract:
Heterotrimeric guanine nucleotide-binding
regulatory proteins (G-proteins) are vital components of numerous
signal transduction pathways, including sensory and hormonal
response systems. G-proteins transduce signals from heptahelical
transmembrane receptors to downstream effectors. The localization
of a G-protein to the plasma membrane, as well as its interaction
with the appropriate receptor and effector, are essential for its
function. In addition, the association of a G-protein's subunits
to form its trimer is required for interaction with its receptor.
The G-protein gamma subunits are subject to a set of
carboxyl-terminal processing events that include prenylation of a cysteine, proteolysis, and
methylation. Recent advances which
elucidate the contributions that the post-translational
modifications of the gamma subunit have on the assembly, membrane
association, and function of the G-protein trimer reveal that
these modifications are required for important protein-protein,
in addition to membrane-protein, interactions.
Assays for G Protein Beta-Gamma Subunit Activity
J.B. Higgins and P.J. Casey
Methods in Neurosciences. Vol. 29,
Chapter 8, pp. 90-100, 1996.
Introduction:
Heterotrimeric guanine nucleotide-binding
regulatory proteins (G proteins) couple transmembrane receptors
to intracellular effectors, resulting in the modulation of second
messenger concentrations. The G protein subunits are designated
alpha, beta, and gamma, in order of decreasing mass, with the
alpha subunit containing the guanine nucleotide-binding site. On
activation, the beta and gamma subunits dissociate from the GTP-bound alpha subunit as a complex. The beta and gamma subunits
do not separate under nondenaturing conditions and therefore the
beta-gamma complex acts functionally as a monomer. The beta-gamma
complex is required for efficient association of the alpha
subunit with the plasma membrane and for activation of the alpha
subunit by the receptor. Additionally, a number of signaling
functions have been directly ascribed to the beta-gamma complex,
the association of beta-gamma with alpha is the most well-defined
protein-protein interaction. One consequence of delineating the
properties associated with alpha/beta-gamma association is that
assays based on these interactions can be used to quantitate
accurately the amount of "active" beta-gamma in a
preparation. In addition, molecules that associate with either
alpha or beta-gamma to disrupt alpha/beta-gamma interactions can
be identified using these types of assays.
The beta-gamma complex modulates the activity of the alpha
subunit by supporting ADP-ribosylation of alpha by pertussis
toxin (PTx) and by decreasing the ability of alpha to exchange
guanine nucleotides, thereby inhibiting the turnover of GTP to
GDP in the GTPase reaction. PTx catalyzes the covalent
modification of certain alpha subunits on a cysteine residue near
the carboxyl terminus, and it is the intact heterotrimer rather
than the free alpha polypeptide that serves as the substrate.
Modification of this particular residue of alpha hinders the
coupling of the heterotrimer to its receptor. A select group of
alpha isotypes (i.e., Gi, Go, Gt)serve as PTx substrates. One
substrate, Go alpha, is particularly abundant in brain,
constituting about 1-2% of the membrane protein in this tissue.
This chapter presents protocols for measuring both the pertussis
toxin-catalyzed ADP-ribosylation of the alpha subunit of Go and
the intrinsic GTPase activity of Go alpha. These two protocols
are used to determine the quantity of active beta-gamma in
samples containing this complex. The sensitivity of the GTPase
assay for beta-gamma is in the low picomole range, whereas the ADP-ribosylation assay is more sensitive and can be used to
quantitate femtomole amounts of beta-gamma. Included are
adaptations of the standard protocols that are useful in
competition studies designed to detect and/or quantitate
molecules that interact with either beta-gamma or Go alpha. The
advantage of these assays is that they measure beta-gamma
accurately reproducibly, and rapidly, and the purification of
both beta-gamma and Go alpha (the G protein subunits required for
the assays) from bovine brain is readily achievable.
In Vitro Processing of
Recombinant G Protein Gamma Subunits: Requirements for Assembly
of an Active Beta-Gamma Complex
J.B. Higgins and P.J. Casey
Journal of Biological Chemistry. 269(12):
9067-9073, 25 March 1996.
Abstract:
The gamma subunits of the heterotrimeric G
proteins are subject to carboxyl-terminal processing. This
processing involves prenylation of a cysteine residue initially 4
amino acids from the carboxyl terminus, endoproteolytic
truncation of the 3 terminal amino acids, and methylation of the
now carboxyl-terminal prenyl-cysteine residue. The significance
of each of these modifications in the ultimate properties of G
proteins is not yet clear. We have developed in vitro
systems for the production of the three processing intermediates
(unmodified, prenylated, and truncated-prenylated) for two gamma
subunits, one which is subject to farnesylation (gamma-1) and one
which is geranylgeranylated (gamma-2). Assessment of the
functional consequences of the processing of gamma was found to
require reconstitution of the polypeptides with a G protein beta
subunit. The ability of recombinant beta, produced in Sf9 cells,
to assemble into stable beta-gamma complexes with each of the
gamma processing intermediates was assessed. Both prenylated and
unprenylated gamma subunits formed stable complexes with beta,
but surprisingly, neither of the truncated-prenylated gamma
subunits were competent for this assembly. The beta-gamma
complexes which were formed were examined for their ability to
interact with a G protein alpha subunit. Only those beta-gamma
complexes containing a prenylated gamma subunit were functional
in this assay. These data indicate that: (1) prenylation of gamma
is not required for beta-gamma assembly; (2) assembly of the
beta-gamma complex occurs prior to the proteolytic processing of
gamma; and (3) beta-gamma complexes require prenylated gamma for
interaction with alpha.
Role of Beta-Gamma Subunits of G Proteins in Targeting
the Beta-Adrenergic Receptor Kinase to Membrane-bound Receptors
J.A. Pitcher, J. Inglese, J.B. Higgins, J.L. Arriza, P.J. Casey,
C. Kim, J.L. Benovic, M.M. Kwatra, M.G. Caron, R.J. Lefkowitz
Science. 257:1264-1267, 28
August 1992.
Abstract:
The rate and extent of the agonist-dependent phosphorylation of
beta2-adrenergic receptors and rhodopsin by beta-adrenergic
receptor kinase (BARK) are markedly enhanced on addition
of G protein beta-gamma subunits. With a model peptide substrate
it was demonstrated that direct activation of the kinase could
not account for this effect. G protein beta-gamma subunits were
shown to interact directly with the COOH-terminal region of BARK,
and formation of the BARK/beta-gamma complex resulted in
receptor-facilitated membrane localization of the enzyme. The
beta-gamma subunits of transducin were less effective at both
enhancing the rate of receptor phosphorylation and binding to the COOH-terminus of BARK, suggesting that the enzyme
preferentially binds specific beta-gamma complexes. The
beta-gamma-mediated membrane localization of BARK serves
to intimately link receptor activation to BARK-mediated
desensitization.
Easter's
Home Page
Chemistry
Department
