[11] - Identification of Putative Direct Effectors for Gαo, Using Yeast Two-Hybrid Method
Introduction
Heterotrimeric G proteins enable the cell to receive and process extracellular signals by linking the G-protein-coupled receptors to intracellular signaling networks.1 When a ligand binds to the receptor a conformational change is induced in the α subunit of the heterotrimeric complex such that the bound GDP is exchanged for GTP. This exchange “activates” the α subunit, which releases the βγ complex resulting in two independent signaling components. The GTP-bound form of the α subunit is able to bind to and regulate downstream signaling components. The identification of direct binding partners for the α subunits has enabled the building of complex signaling networks that control many aspect of cellular function.2
To identify effectors for the α subunit of the Go heterotrimeric G protein, a yeast two-hybrid strategy was utilized.3,4 The constitutively activated form of the αo subunit was used in the screen. This form is constitutively in the activated state because of a mutation that abolishes the intrinsic GTPase activity and therefore the α subunit is bound preferentially to GTP versus GDP. Because this is the activated state of the G protein α subunit it should preferentially interact with direct downstream signaling molecules. We used an embryonic day 12 chick dorsal root ganglion (DRG) library that we had constructed to carry out the screen.5 Here we describe the methods, depicted as a flow chart in Fig. 1, used to identify putative effectors for the Gαo subunit.
Section snippets
Materials
The yeast two-hybrid system is available from Life Technologies (Rockville, MD) as the ProQuest two-hybrid system.4 All media including YPD (yeast extract–peptone–dextrose) and SD (synthetic defined) dropout reagents are obtained from Clontech (Palo Alto, CA). Primers are synthesized by MWG Biotech (High Point, NC).
Small-Scale Yeast Transformation
To perform the two-hybrid control reactions the bait and library plasmid must be introduced into the yeast strain MaV203.6,7 This is done by transforming the yeast strain by the lithium acetate method.8 The yeast strain MaV203 is streaked onto a YPD plate and incubated at 30° for 48 hr. Several colonies of yeast are suspended in 100 μl of sterile 0.9% (w/v) saline solution and spread onto a fresh YPD plate in a 2 × 2 cm patch. The plate is incubated overnight at 30°. The patch of yeast
Construction of Two-Hybrid Bait
The constitutively activated α subunit of Go (rat species) is amplified by polymerase chain reaction (PCR), using Pfu polymerase and the following primers: 5′-ACGTGTCGACCATGGGATGTACTCTGAGC-3′ and 5′-AGCTTAGCGGCCGCTCAGTACAAGCCACAGCC-3′. These primers encode a SalI restriction site and a NotI restriction site, respectively. The PCR product and two-hybrid bait vector pDBLeu (Life Technologies) are digested with SalI and NotI and gel purified. The α subunit is subcloned into the bait vector and
Yeast Two-Hybrid Controls
Once the construct expression is verified it is necessary to perform control reactions to determine the amount of leakiness of the histidine (His) reporter as well as whether there is any autonomous activation of the other reporter genes. For these controls yeast are transformed with either the empty pDBLeu vector or pDBLeu- plus the empty library vector pPC86, plated on SD/–Leu–Trp dropout medium, and grown for 2–3 days at 30°. To determine the correct 3-amino-1, 2,
Library Screening
Once it has been determined that there is no autonomous reporter gene activation the library screening can begin. A chick neuronal library has already been prepared and cloned into the yeast GAL4 activation domain plasmid pPC86. Pre-made competent yeast strain MaV203 (Life Technologies) is used. The PEG–LiAc solution is thawed out at room temperature and mixed well. The competent cells are thawed by placing them in a 30° water bath for 1 min. Five hundred microliters of cells is aliquoted into
Characterization of Positive Clones
Once positive clones have been obtained it is necessary to characterize the reporter gene activation. Clones that grow on the initial library screen are streaked onto fresh SD/–Leu–Trp plates and grown at 30° for 2 days. Three individual colonies for each positive clone are then streaked onto an SD/–Leu–Trp plate and incubated for 18–20 hr. The streaked plate is then replica plated onto a YPD plate containing a nitrocellulose membrane as well as an SD/–Leu–Trp–His plus 3-AT and an
Isolation of Plasmid DNA Encoding Putative Interacting Proteins
It is necessary to isolate the plasmid DNA specific for the pPC86 library clones obtained in the library screening.9 A colony of each candidate clone is inoculated into 2 ml of SD/–Leu–Trp and grown overnight until saturation at 30° with shaking. The yeast cells are then pelleted by centrifugation in a microcentrifuge tube at 13,000g for 2 min at room temperature. The medium is removed and the pellet is resuspended in 100 μl of lysis buffer [2% (w/v) SDS, 0.2 N NaOH] and incubated for 10 min at
Confirmation of Putative Interacting Proteins
Once candidate plasmids have been isolated it is necessary to reintroduce them into the yeast strain to determine whether the reporter gene activation is dependent on the interaction with the bait. This is done by introducing the pPC86 candidate plasmid with either the pDBLeu vector only or the pDBLeu- construct. The transformation is done using the small-scale transformation. The yeast is plated onto SD/–Leu–Trp and independent clones are isolated. These clones are then
Specificity of Interactions
Once it has been shown that the interaction is dependent on the presence of the subunit further experiments can be done to determine the specificity of the interaction. The first set of experiments is to determine whether the interaction is specific for the activated form of the αo subunit that is mimicked by the constitutively activated form of αo. This is done by transforming the yeast strain with the pPC86 library clone with either the wild-type or constitutively activated form of αo
Two-Hybrid Clone Identification and Independent Confirmation
Once clones have been confirmed by the reintroduction of bait and prey plasmids into the yeast it is necessary to determine the sequence identification of the clones. This can be done by sequencing the minipreparation plasmid DNA obtained after the electroporation and isolation step in the above-described confirmation assay. The sequencing primer used is within the GAL4 activation domain about 100 bp upstream of the SalI insert site. The primer sequence used is 5′-AACGCGTTTGGAATCACT-3′ and
Results
We screened an embryonic d00ay 12 chick DRG library with the constitutively activated form of Gαo. The system that we used is now available from Life Technologies as the ProQuest two-hybrid system. This system has the benefit of having few false positives when used with the MaV203 yeast strain. In our screen we had only one false positive after screening 5 × 106 clones. The positive clones isolated are listed in Table I. Several of these clones were isolated independently of our laboratory,
Concluding Remarks
With the identification of additional Gα-interacting proteins the role that G proteins play in signal transduction will inevitably increase, as will the complexity of signaling in general. The yeast two-hybrid method aids in this process by providing a powerful tool to help elucidate the roles that G proteins play in signaling by enabling the identification of novel G protein-interacting proteins.
Acknowledgments
Research in our laboratories is supported by NIH Grants GM-54508 and DK-38761 to R.I. J.D.J. is supported by the MSTP and currently by an individual predoctoral fellowship (DA-05798).
References (12)
- et al.
Cell
(2000) - et al.
J. Biol. Chem.
(1999) - et al.
J. Biol. Chem.
(1999) - et al.
J. Biol. Chem.
(1999) Annu. Rev. Biochem.
(1987)- et al.
Nature (London)
(1989)
Cited by (1)
Identification and biochemical analysis of GRIN1 and GRIN2
2004, Methods in Enzymology