G Protein
Chimera Users Manual
Introduction
Since the
first description of G protein chimeras that can alter the signaling phenotype
of receptors, many investigators have found them useful for a variety
of research purposes. Several people who work with Gi-coupled
receptors have found that it is easier to study the stimulation of phospholipase
C than the inhibition of adenylyl cyclase. Several groups have used the
chimeras to develop rapid assays of receptor activation that can be used
for screening mutants or agonist drugs. Others have used the chimeras
to complement mutant receptors in detailed structure-function studies.
Over the past four years, I have sent over 200 samples of chimeras. The collection
of chimeras has gradually grown and has been improved by the addition
of epitope-tagged versions. This update should help people use the chimeras
most effectively. Many people who received the original clones may want
to upgrade to the new versions.
Structure-Function Studies with G Proteins
The carboxyl-terminus of the G alpha protein subunit is a key determinant of receptor specificity.
We have previously shown that the Gq alpha subunit (alpha q) can be made
to respond to Gi alpha-coupled receptors by replacing its carboxyl-terminus
with the corresponding Gi2 alpha, Go alpha, or Gz alpha residues (2).
We have recently extended these findings in three ways:
- C-terminal mutations of Gq alpha/Gi alpha chimeras show that the critical amino
acids are in the -3 and -4 positions.
- Exchange of carboxyl-termini between Gq alpha and Gs alpha allows activation
by receptors appropriate to the C-terminal residues.
- We identify receptors that either do or do not activate the expected C-terminal
chimeras (Gq alpha/Gi alpha, Gq alpha/Gs alpha, Gs alpha/Gq alpha).
Replacement of the five carboxyl-terminal amino acids of Gq alpha with the Gs alpha
sequence permitted a Gs alpha-coupled receptor (the V2 vasopressin receptor,
but not the beta 2-adrenoceptor) to stimulate phospholipase C. Replacementof
the five carboxyl-terminal amino acids of Gs alpha with residues of Gq
alpha permitted certain Gq alpha-coupled receptors (bombesin and V1a vasopressin
receptors, but not the Oxytocin receptor) to stimulate adenylyl cyclase.
Thus, the relative importance of the G alpha carboxyl-terminus for permitting
coupling to a new receptor depends on the receptor with which it is paired.
These studies refine our understanding of the basis of receptor-G alpha
specificity. Substitutions of the C-termini of Gq alpha and other G-alpha
subunits has recently been instrumental in developing high throughput
screens for new agonists of G protein-coupled receptors [Broach J.R. and
Thorner J. (1996) High-throughput screening for drug discovery. Nature
384 (Suppl.):14-16].
Chimera Summary
Notes on the chimeras:
- All have been subcloned into pcDNA-1, in the Bam HI/NsiI cassette with q4WT as
parent construct for the "q" chimeras and Gs-WT-HA as the
parent construct for the "s"chimeras (see below for the description
of the parent constructs).
- All have the internal HA epitope, which does not affect receptor coupling, yet
allows recognition by the 12CA5 antibody (available from Boehringer
Mannheim as a purified monoclonal and directly conjugated to HRP, which
is convenient for Westerns).
- All the constructs are in pcDNA-1, which require sup F selectionfor Amp and
Tet resistance. This requires special competent bacteria that are available
in most labs, but can also be purchased from Invitrogen (for example,
mc1061/p3).
- qi5-HA: This is Gq alpha with the C-terminal amino acids changed from Gq alpha
to Gi alpha residues (EYNLV to DCGLF). This construct allows many Gi-coupled
receptors to stimulate phospholipase C (PLC). This is the most popular
chimera, perhaps because it is easier to talk about coupling to Gi-coupled
receptors with "qi5" rather than "qo5" or "qz5."
Click
here to see sequence for qi5.
- qo5-HA:
This is Gq alpha with the C-terminal amino acids changed from Gq alpha
to Go alpha residues (EYNLV to GCGLY). Works the same as qi5 but (for
unknown reasons) has a slightly lower basal PLC activity. This can increase
the signal-to-noise ratio, so I tend to use it the most. Click
here to see sequence for qo5.
- qz5-HA:
This is Gq alpha with the C-terminal amino acids changed from Gq alpha
to Gz alpha residues (EYNLV to YIGLC). Works the same as qi5 and is
the least popular since no one knows what Gz alpha really does in nature.
Since qz5 is not sensitive to pertussis toxin, qz5 may be the only G
protein activated by a Gi-coupled receptor in cells treated with pertussis
toxin. This trick can be experimentally useful in settings where you
want experimental control of the exact G protein and the receptor that
is activated. It is theoretically possible that this construct will
work better than the other constructs for particular receptors, but
I have not seen this happen yet.
- qs5-HA:
This is Gq alpha with the C-terminal amino acids changed from Gq alpha
to Gs alpha residues (EYNLV to QYELL). This construct allows some Gs-coupled
receptors to stimulate phospholipase C. Click
here to see sequence for qs5.
- sq5-HA:
This is Gs alpha with the C-terminal amino acids changed from Gs alpha
to Gq alpha residues (QYELL to EYNLV). This construct allows some Gq-coupled
receptors to stimulate Adenylate cyclase. There isn't much experience
with this chimera at the moment. It may be useful for people who find
the AC stimulation is better readout of receptor activation than PLC
stimulation.
- 13Z:
Note: This construct is only available from Dr. Tatyana Voyno-Yasenetskaya. This is G13 alpha with the C-terminal amino acids changed from G13 alpha
to Gz alpha residues (QLMLQ to YIGLC). This construct allows some Gi-coupled
receptors to stimulate an increase in pH of cells. There isn't much
experience with this chimera at the moment, but it was used successfully
with the D2-dopamine receptor [see Voyno-Yasenetskaya et al. (1994)
JBC 269:4721-4724]. Please note
that this construct is not epitope tagged since no one has made a reliable
internal tag for G13 alpha.
Parent Constructs:
- q4WT:
This is Gq alpha with an HA epitope engineered into an internal site
that does not seem to affect receptor coupling in multiple studies.
Epitope tagged by Paul Wilson, see Wedegaertner, JBC 268:
25001-25008. The 5" non-coding sequences were removed, but the
3' non-coding sequences remain, as in Strathmann & Simon (1990)
PNAS 87:9113-9117. The parent construct has been donated
to the ATCC by the Bourne
Lab.
Click here to see sequence for
q4WT
- Gs-WT-HA:
This construct is also known as "GSL" in the Bourne Lab.
This is "wild type" Gs alpha in pcDNA-1 with a HA epitope
engineered into an internal site that does not seem to affect receptor
coupling in multiple studies. [See Levis & Bourne (1992) J. Cell
Biol. 119:1297-1307] The parent construct has been donated
to the ATCC by the Bourne
Lab.
Click here to see sequence
for Gs-WT-HA.
Below is
a list of publications that describes how we have used the G alpha C-terminal
chimeras:
Initial
description of chimeras:
- Conklin
B.R., Farfel Z., Lustig K.D., Julius D. and Bourne H.R. (1993) Substitution
of three amino acids switches receptor specificity of Gq alpha to that
of Gi alpha. Nature 363:274-276.
- Conklin
B.R., Herzmark P., Ishida S., Voyno-Yasenetskaya T.A., Sun Y. and Bourne
H.R. (1996) C-Terminal mutations of Gq alpha and Gs alpha that alter
the fidelity of receptor activation. Mol.Pharmacol. 50:885-890.
- Voyno-Yasenetskaya
T., Conklin B.R., Gilbert R.L., Hooley R., Bourne H.R. and Barber D.L.
(1994) G13 alpha stimulates Na-H Exchange.J. Biol. Chem. 269:4721-4724.
Chimeras
used in studies from 1993–1999 (over 50 publications have used the G protein chimeras and I have stopped keeping track):
- Liu J.,
Conklin B.R., Blin N., Yun J., Wess J. (1995) Identification of a receptor/G-protein
contact site critical for signaling specificity and G-protein activation.
Proceedings of the National Academy of Sciences, U.S.A. 92:11642-11646.
- Messier
T.L., Dorman C.M., Bräuner-Osborne H., Eubanks D. and Brann M.R.
(1995 ) High throughput assays of cloned adrenergic, muscarinic, neurokinin,
and neurotrophin receptors in living mammalian cells. Pharmacol.
Toxicol. 76:308-311.
- Liu J.,
Blin N., Conklin B.R. and Wess J. (1996) Molecular mechanisms involved
in muscarinic acetylcholine receptor-mediated G protein activation studied
by insertion mutagenesis. J. Biol.Chem. 271:6172-6178.
- Boss V.,
Talpade D.J. and Murphy T.J. (1996) Induction of NFAT-mediated transcription
by Gq-coupled receptors in lymphoid and non-lymphoid cells. J. Biol.
Chem. 271:10429-10432.
- Arai H.
and Charo I.F. (1996) Differential regulation of G-protein-mediated
signaling by chemokine receptors. J. Biol. Chem. 271:21814-21819.
- Liu J.,
Blin N., Conklin B.R. and Wess J. (1996) Molecular mechanisms involved
in muscarinic acetylcholine receptor-mediatedG protein activation studied
by insertion mutagenesis, J. Biol.Chem. 271:6172-6178.
- Gomeza
J., Mary S., Brabet I., Parmentier M.-L, Restituito S., Bockaert J.
and Pin J.-P. (1996) Coupling of metabotropic glutamate receptors 2
and 4 to G15 alpha, G16 alpha, and chimeric Gqalpha/Gi alpha proteins:
Characterization of new antagonists. Mol. Pharmacol. 50:923-930.
- Parmentier
M.-L., Pin J.-P., Bockaert J. and Grau Y. (1996) Cloning and functional
expression of a drosophila metabotropic glutamate receptor expressed
in the embryonic central nervous system. J. Neurosci. 16:6687-6694.
- Burstein
E.S., Bräuner-Osborne H., Spalding T.A., Conklin B.R. and Brann
M.R. (1997) Interactions of muscarinic receptors with the heterotrimeric
G proteins Gq and G12: Transduction of proliferative signals. J.
Neurochem. 68:525-533.
- Kostenis
E., Gomeza J., Lerche C. and Wess J. (1997) Genetic analysis of receptor-Gaq
coupling selectivity. J. Biol. Chem.272:23675-37681.
- Monteclaro
F.S., Arai H. and Charo I.F. (1997) Molecular approached to identifying
ligand binding and signaling domains of c-c chemokine receptors. Methods
Enzymol. 288:70-84.
- Tsu R.C.,
Ho M.K.C., Yung L.Y., Joshi S. and Wong Y.H.(1997) Role of amino- and
carboxyl-terminal regions of Gaz inthe recognition
of Gi-coupled receptors. Mol. Pharmacol.52:38-45.
- Coward
P., Wada H.G., Falk M.S., Chan S.D.H., Meng F., Akil H. and Conklin
B.R. (1998) Controlling signaling with a specifically designed Gi-coupled
receptor. Proc.Natl. Acad.
Sci. 95:352-357.
- Ancellin,
N., and Hla, T. (1999) Differential pharmacological properties and signal
transduction of the sphingosine 1-phosphatereceptors EDG-1, EDG-3, and
EDG-5. J. Biol. Chem. 274:18997–19002.
I hope this
information is useful. Feel free to contact me if you have suggestions.
Mailing
Address:
Bruce
R. Conklin, M.D.
The Gladstone
Institute of Cardiovascular Disease
Departments
of Medicine and Pharmacology, UCSF
1650 Owens Street
San Francisco,
CA 94158
Other
information:
Office:
(415) 734-2712
Administrative Assistant: (415)
734-2716
Fax: (415)
355-0960
bconklin@gladstone.ucsf.edu
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Content
last modified: April 2004
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