Biology Reference
In-Depth Information
Abstract
Microtubules, major components of the cytoskeleton, play important roles in a va-
riety of cellular functions including mitosis, intracellular transport, and the modula-
tion of cell morphology. Several studies have demonstrated that specific G-protein
alpha subunits bind to tubulin with a high affinity (
130 nM) and elicit various func-
tional effects on tubulin and microtubules. In this chapter, we present a description of
the protocols for several methods that are used to determine the interaction between
heterotrimeric G proteins and tubulin, as well as functional consequences of the in-
teractions including protocols for protein purification, binding assays, tubulin
GTPase assays, microtubule dynamics assays, and assays for cytoskeletal conse-
quences of G-protein-coupled receptor signaling.
INTRODUCTION
Microtubules have been implicated inG-protein signaling for more than 30 years, and a
direct binding between select G-protein
a
subunits (Gs
a
,Gi1
a
,andGi2
a
) and tubulin
was observeda decade later (
Wang,Yan,&Rasenick, 1990
). Thiswas followedbydem-
onstration of tubulin interaction with Gq
a
for regulation of phospholipase signaling
(
Popova, Garrison, Rhee,&Rasenick, 1997
). Initial studies suggested thatmicrotubules
were instrumental in G-protein signaling and that their disruption facilitated signaling,
by liberatingGproteins fromsome unidentified constraint. Subsequent studies revealed
direct transactivation of G
a
subunits due to transfer of GTP between tubulin and G
a
(
Popova et al., 1997;Yan, Popova,Moss, Shah,&Rasenick, 2001
), althougha structural
basis for this was not revealed until recently (
Dave et al., 2011; Layden et al., 2008
).
During the course of these studies, it became apparent that the interactions between
tubulin andG
a
were bidirectional and that G
a
was capable of activating tubulinGTPase
(
Roychowdhury & Rasenick, 1994
) and this increased dynamic behavior of microtu-
bules (
Roychowdhury, Panda, Wilson, & Rasenick, 1999
). While Gs
a
had long been
observed in the cytosol, lending credibility to the possibility that it could regulatemicro-
tubules (
Rasenicket al., 1984
), thedemonstration that this accompanied the activationof
Gs
a
(
Wedegaertner, Bourne, &vonZastrow, 1996
) and the observation of this phenom-
enon in living cells (
Yu & Rasenick, 2002
) gave rise to the observations that activated
Gs
a
could, indeed, regulate microtubule-based neurite outgrowth (
Yu, Dave, Allen,
Sarma, &Rasenick, 2009
) and microtubule dynamics (
Dave et al., 2011
). These obser-
vations and themethods thatmade thempossiblewill constitute the basis of this chapter.
12.1
GENERAL PROTOCOLS
12.1.1
In vitro determination of the association between tubulin
and Gs
a
Several
in vitro
binding assays including an overlay method using
125
I tubulin and G
proteins immobilized nitrocellulose (
Wang & Rasenick, 1991
), a pull-down assay
(
Yu et al., 2009
), and surface plasmon resonance (SPR) (
Dave et al., 2011
) have been
