A molecular and structural mechanism for G protein-mediated microtubule destabilization.

A Molecular and Structural Mechanism for G-protein Mediated
Microtubule Destabilization
Rahul H. Davé1, 3, Witchuda Saengsawang1, Manu Lopus4, Sonya Davé5, Leslie Wilson4 and
Mark and M. Rasenick1, 2
From 1Department of Physiology and Biophysics, 2Psychiatry, 3MSTP program, University of Illinois at Chicago, Chicago, Illinois 60612 4 Department of Molecular, Cellular,
and Developmental Biology and the Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA 93106 5Department of Molecular
Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37212
J Biol Chem. 2010 (in press)
Poster by Andrej Bieri
Introduction
Results
Conclusion
● Heterotrimeric guanine nucleotide binding proteins2 (G proteins) are responsible for inducing intracellular signaling cascades in response to hormone or neurotransmitter
activation of G-protein coupled receptors (GPCRs). Conformational changes of the GPCRs are leading to the exchange of GDP for GTP on the G α subunits and its
dissociation from the Gβγ dimer and the receptor.
● Gs α is interacting with adenylyl cyclase which generates cyclic AMP from ATP. Moreover, active Gs α, independently of cAMP, can directly bind to microtubules and
promotes its dynamic instability by stimulating tubulin GTPase.
● Microtubules are polymers composed of alpha:beta tubulin dimers. When the GTP in the beta subunit is exchanged with GDP, the subunit is prone to depolimerization.
Thus, the activation of tubulin GTPase by Gs α increases microtubule dynamics. This is suggesting an important role in hormone or neurotransmitter induced regulation of
cellular morphology.
● Here, the role of the α3-β5 region of Gs α in microtubule destabilization is reported and molecular modeling studies suggest the action of Gs α/tubulin interface at the
microtubule plus end.
Tubulin Binding
Binding and Kinetics of Gs α-Tubulin Complexes
The G227L mutation of Gs α (GsαQL ) remains
constitutively bound to GTP and is therefore active. It
bound tubulin with an affinity of Kd = 100 nm whereas
inactive Gs α-GDP did not bind.
➔ Gs α must be active in order to bind tubulin.
Peptides from the α3-β5 Region Bind to Tubulin
Following table shows the sequence of peptides
derived from the α3-β5 region of Gs α and Gt α and its
affinities for tubulin. Gt α was used as control since it
does not bind tubulin.
Region near
N-ter of Gα
15AA of the
α3-β5 region
Derivative
peptides in which
some residues
were replaced
by their Gtα
homologues
➔ None of the Gt α-derived peptides bound tubulin
➔ Peptides derived from Gs α bound tubulin.
➔ M2 bound tubulin similar to P3.
➔ Other derivative peptides either didn't bind tubulin
or only with low affinity.
PN KQLQKDKQVYRATHR Kd = 10 μM
PGtN EDAEKDARVYRATVK No binding
P3 LNLFKSIWNNRWLRT Kd = 40 μM
PGt3 LHLFNSICNHRYFAT No binding
M1 LHLFN SIWNNRWLRT Kd = 373 μM
M2 LNLFKSICNH RWLRT Kd = 45 µM
M3 LNLFKSIWNNRYFA T Kd = 313 μM
M5 LHLFNSIWNNRYFA T No binding
Tubulin GTPase Activation
Gs α Activation of Tubulin GTPase is Unaltered by
Mutating the α3-β5 Loop
A chimera of Gs α and Gt α (GsαGtL/QL ) showed the
functional importance of the α3-β5 loop.
➔ GsαQL stimulated tubulin
GTPase with an EC 50 of
1.2 μM Gsα.
➔ GsαGtL/QL mutants exhibited
greater activation of
tubulin GTPase activity.
➔ GsαGtL/QL bound tubulin
Similarly to parent GsαQL.
➔ Importance of α3-β5 loop is diminished in context
of to the whole protein.
α3 helix α3-β5 Loop β5 sheet
Gs α (276-282) NRLQEALNLFKSI WNNRWLR TISVILF
Gtα NRMHESLHLFNSI CNHRYFA TTSIVLF
GsαGtL/QL (276-282) NRLQEALNLFKSI CNHRYFA TISVILF
Gs α-derived Peptides Functionally Mimic the
G-protein Stimulation of Tubulin GTPase
The effect of the short peptides from tubulin binding
studies was tested.
➔ P3 EC50 = 24 μM
➔ M2 EC50 = 47 μM
➔ M1, M5 and peptides from N-ter failed to stimulate
tubulin GTPase
➔ P3 and M2, peptides corresponding to the α3-β5
region of Gs α, mimic the effect of the entire protein.
Effect on Microtubule Dynamics
The Active Conformation of Gs α Increases
Microtubule Dynamic Instability
Active GsαQL destabilized
microtubules in a concentration
dependent manner with an
IC50 of 3.5 ± 0.4 μM.
GsαQL increased the growing rate, the shortening
rate and the catastrophy frequency.
➔ Growing rate: +25 % (1 μM) and +63 % (2 μM)
➔ Catastrophy frequency: +73 % (2 μM)
➔ Dynamicity: +42 % (1 μM) and +63 % (2 μM)
GsαWT had no significant effect on any dynamics
parameter.
GsαGtL/QL promote microtubule depolymerization
more strongly than GsαQL . This is consistent with its
stronger effect on tubulin GTPase.
➔ Growing rate: +94 % (2 μM)
➔ Catastrophy frequency: +138 % (2 μM)
➔ Dynamicity: +104 % (2 μM)
Gs α-derived Peptides Increase Microtubule
Dynamics
The α3-β5 derived peptide P3 increased microtubule
dynamic but required higher concentrations.
➔ Growing rate: +63 % (20 μM)
➔ Catastrophy frequency: +188 % (20 μM)
➔ Dynamicity: +68 % (20 μM)

● The data presented in this paper suggest a model for the action of Gs α on microtubules.
2
● Gs α promotes hydrolysis of GTP on tubulin and sequesters the newly released tubulin-GDP. This is resulting in increased micrutubule dynamics.
● Gs α must be in an active conformation in order to bind tubulin, to stimulate tubulin GTPase and to increase microtubule dynamic instability.
● 1 μ M Gs α causes a two-fold increase in the catastrophe frequency with minimal depolymerization of the microtubules. This suggests that the primary function of Gs α may
be to increase microtubule dynamics rather than affect the mass of assembled polymer.
● The α3-β5 region appears to be the principal region of tubulin GTPase and is may be more important than the loop itself. Mutagenesis of this region alters Gs α
stimulation of tubulin GTPase and microtubule dynamics.
● Short peptides were developed that mimic the effect of Gs α on tubulin and microtubules (P3, M2, M3) and that only bind tubulin without effect on he microtubule
dynamics (M1, PN). These peptides should be useful tools to probe Gs α effect in living cells.
● Finally, this report suggest a mechanism for neurotransmitter-induced remodeling of the cytoskeleton. Furthermore, it rase the possibility that small-molecule probes can
be generated to manipulate this process.