Interactions of the C Terminus of Metabotropic Glutamate
Receptor Type 1
␣
with Rat Brain Proteins: Evidence for a
Direct Interaction with Tubulin
F. Ciruela, M. J. Robbins, *A. C. Willis, and R. A. J. McIlhinney
Medical Research Council Anatomical Neuropharmacology and *Immunochemistry Units, Oxford, England
Abstract: Metabotropic glutamate receptors (mGluRs)
are coupled to G protein second messenger pathways
and modulate glutamate neurotransmission in the brain,
where they are targeted to specific synaptic locations. As
part of a strategy for defining the mechanisms for the
specific targeting of mGluR1
␣
, rat brain proteins which
interact with the intracellular carboxy terminus of
mGluR1
␣
have been characterized, using affinity chro-
matography on a glutathione S-transferase fusion protein
that contains the last 86 amino acids of mGluR1
␣
. Three
of the proteins specifically eluted from the affinity column
yielded protein sequences, two of which were identified
as glyceraldehyde-3-phosphate dehydrogenase and

-tubulin; the other was an unknown protein. The identity
of tubulin was confirmed by western immunoblotting.
Using a solid-phase binding assay, the mGluR1
␣
–tubulin
interaction was shown to be direct, specific, and satura-
ble with a K
D
of 2.3 Ϯ 0.4
M. In addition, mGluR1
␣
, but
not mGluR2/3 or mGluR4, could be coimmunoprecipi-
tated from solubilized brain extracts with tubulin using
anti-

-tubulin antibodies. However, mGluR1
␣
could not
be coimmunoprecipitated with the tubulin binding protein
gephyrin, nor could it be coimmunoprecipitated with
PSD95. Collectively these data demonstrate that the
last 86 amino acids of the carboxyl-terminal tail of
mGluR1
␣
are sufficient to determine its interaction with
tubulin and that there is an association of this receptor
with tubulin in rat brain. Key Words: Metabotropic—
Glutamate receptor—Tubulin—Targeting.
J. Neurochem. 72, 346 –354 (1999).
Metabotropic glutamate receptors (mGluRs) constitute
a family of large G protein-coupled receptors that show
little sequence homology with the superfamily of smaller
G protein-linked receptors (Tanabe et al., 1992). Eight
members of the mGluR family have been identified,
several of which generate different subtypes by alterna-
tive mRNA splicing (Knopfel et al., 1995; Pin and Du-
voisin, 1995). These eight mGluR receptors and their
subtypes may be categorized into three subgroups on the
basis of their sequence homology, agonist selectivity,
and signal transduction pathway. The first group contains
mGluR1 and mGluR5, which share the strongest se-
quence homology (62% identical at the amino acid lev-
el), are coupled to phospholipase C in transfected cells,
and have quisqualic acid as their most potent agonist.
The second group consists of mGluR2 and mGluR3,
which couple negatively to adenyl cyclase in transfected
cells and for which
L
-2-(carboxycyclopropyl) glycine is
the most potent agonist. The third group contains
mGluR4, mGluR6, mGluR7, and mGluR8, which also
couple negatively to adenyl cyclase but have
L
-2-amino-
4-phosphonobutyric acid as their most potent agonist.
Splice variants for mGluR1, mGluR4, mGluR5, and
mGluR7 have been described, which involve alterations
in their C-terminal sequences (Pin et al., 1992; Simoncini
et al., 1993; Minakami et al., 1994; Flor et al., 1997).
Four C-terminal splice variants of mGluR1 have been
described, namely, mGluR1
␣
, mGluR1

, mGluR1c, and
mGluR1d (Pin et al., 1992; Knopfel et al., 1995). In
mGluR1

, the insertion of an 85-base sequence in the
carboxy-terminal tail introduces a stop codon that results
in a 318-amino acid deletion and also changes the read-
ing frame, resulting in a different and much shortened
C-terminal amino acid sequence from that of mGluR1
␣
.
Similarly, in mGluR1c and mGluR1d a different inser-
tion at the same position results in further changes in the
sequence of the C-terminal tails, which are also much
smaller than that of mGluR1
␣
. An additional splice
variant of mGluR1, mGluR1e, is known that appears to
result in the production of the N terminus of the protein
only (Pin and Duvoisin, 1995).
Received June 5, 1998; revised manuscript received July 29, 1998;
accepted July 29, 1998.
Address correspondence and reprint requests to Dr. R. A. J. McIl-
hinney at MRC Anatomical Neuropharmacology Unit, Mansfield Road,
Oxford OX1 3TH, U.K.
Abbreviations used: BSA, bovine serum albumin; GAPDH, glycer-
aldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase;
mGluR, metabotropic glutamate receptor; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; PBS-Ca/Mg, phos-
phate-buffered saline containing 0.8 mM CaCl
2
and 0.4 mM MgCl
2
;
PBS-N, phosphate-buffered saline containing 0.05% Nonidet P-40;
PSD, postsynaptic density; PVDF, polyvinylidene difluoride; SDS,
sodium dodecyl sulphate.
346
Journal of Neurochemistry
Lippincott Williams & Wilkins, Philadelphia
© 1999 International Society for Neurochemistry
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