27 - GoLoco Motif Peptides as Probes of Gα Subunit Specificity in Coupling of G-Protein-Coupled Receptors to Ion Channels
Introduction
The remarkable specificity of signaling pathways linking the G-protein-coupled superfamily of receptors (GPCRs) to a vast array of effector proteins is commonly thought to be related to the large number of molecular species directly in the signaling pathways, those modulating the pathways indirectly, and those providing compartmentalization of elements of the pathways. There is ample evidence for subclasses of signaling cascades largely segregated by the class of G-protein α subunit involved (e.g., Gs, Gi/o, Gq/11). However, despite some circumstantial evidence that specificity in a given signaling cascade in each subclass resides in one or more of these elements, little experimental evidence exists linking a particular GPCR effector pathway to specific cascade elements such as specific Gα, Gβ, or Gγ subunits. For example, the coupling of GPCRs through Gi/o proteins to effectors such as GIRK and calcium channels occurs through Gβγ subunits (Herlitze 1996, Ikeda 1996, Logothetis 1987) but is largely independent of the particular Gβ or Gγ subtype (Wickman et al., 1994; but see Kleuss 1992, Kleuss 1993). Evidence for Gα subunit specificity has to this point derived from three types of experiments: reconstitution of signaling blocked by pertussis toxin using intracellular dialysis of purified or recombinant Gα subunits (Degtiar 1997, Ewald 1989; but see McGehee and Oxford, 1991), block of function with Gα-specific antibodies (Menon-Johansson 1993, Takano 1997), maintenance of signaling under PTX treatment by expression of mutant Pertussis toxin (PTX)-resistant Gαi/o subunits (Kammermeier 2003, Leaney 2000; see also Chen 2004, Ikeda 2004), or the absence of signaling during the antisense knockdown of Gαi/o subunits (Degtiar 1996, Delmas 1999, Kleuss 1991, Takano 1997). All of these approaches, however, involve either overexpression of a Gα subunit, which alters normal signaling stoichiometry, or incomplete reduction in specific Gα subunit expression with the attendant ambiguity of sufficient amplification for signaling from the remaining subunits of that species.
Regulator of G-protein signaling (RGS) proteins are a large family of multidomain proteins that modulate GPCR signaling in a variety of ways. The namesake domain of this family, the RGS box, has been shown to accelerate the intrinsic GTPase activity of certain Gα subunits and termination of signaling. Another 19 amino acid domain, found in several RGS proteins, as well as other unrelated proteins, is the Gαi/o–Loco interaction or GoLoco motif (Siderovski et al., 1999). We have taken advantage of three features of the GoLoco motifs found in RGS12, RGS14, and AGS3 proteins to develop an alternative approach to defining GPCR-to-G-protein coupling specificity. The key features of these particular GoLoco motifs are their guanine nucleotide dissociation inhibitor (GDI) activity, which essentially locks Gαi subunits in their inactive GDP-bound form; their binding to a site on Gαi that precludes association of the cognate Gβγ subunits, leaving Gαi in its monomeric inactive form; and the unique specificity of this binding for Gαi but not Gαo proteins (Kimple 2001, Kimple 2002). These features suggest the possibility that synthetic peptides encoding these GoLoco motifs could interrupt signaling in pathways preferentially utilizing Gαi over Gαo subunits, thus discriminating signaling specificity at this level. Such peptides would represent relatively small, but specific probes of the Gαi–Gβγ interface, reducing ambiguity associated with overexpression of signaling components or with specificity of antibody block. By perfusing synthetic GoLoco motif peptides into AtT20 cells, we have demonstrated interference with the coupling of D2 receptors to Kir3.1/3.2 channels and the absence of such interference in somatostatin (SST) receptor coupling to these channels and to D2 receptor coupling to P/Q-type calcium channels in the same cells, suggesting differential Gα subunit signaling for each pathway.
Section snippets
Selection and Synthesis of GoLoco Motif Peptides
While GoLoco motifs have been found in a variety of proteins, those that bind Gαi subunits, but not Gαo subunits, preferentially are found in RGS12, RGS14, and AGS3. Thus it was decided to synthesize GoLoco motif peptides derived from RGS12 (“R12GL,” EAEEFFELISKAQSNRADDQRGLLRKEDLVLPEFLR; amino acids 1186–1221 of SwissProt accession number O08774) and RGS14 (“R14GL,” DIEGLVELLNRVQSSGAHDQRGLLRKEDLVLPEFLQ; amino acids 496–531 of SwissProt accession number O08773) representing the minimal fragment
Choice of Cell for Evaluation of GPCR-to-Ion Channel Coupling
A variety of cells and cell lines have been employed in the study of GPCR signaling. In the case of GPCR-to-GIRK channel coupling, most studies have utilized native tissues (e.g., atrial muscle cells or neurons) with a fixed complement of all signaling partners, heterologous expression of receptors and channels in a “null background” cell [e.g., Xenopus oocytes, Chinese hamster ovary (CHO) cells, or human embryonic kidney (HEK) cells], or cell lines natively expressing some or all components.
Cell Culture, Plating, and Transfection Procedures
Our culture procedures for AtT20 cells involve propagating and maintaining them in Ham's F10 culture medium supplemented with 5% fetal bovine serum, 10% heat-inactivated horse serum, 2 mM l-glutamine, and 1 mg/ml gentamycin antibiotic. Cells are maintained at 37° in 5% CO2 in 25-cm plastic tissue culture flasks and passaged every 4 to 5 days at a 1:5 dilution. With this regimen, cells do not reach confluence in the flasks; rather, they occupy roughly 50% of the surface. A standard trypsin-EDTA
Electrophysiological Methods to Measure GIRK Channel Currents
We employ standard whole cell patch-clamp methods (Hamill et al., 1981) to record agonist-induced GIRK or calcium channel currents from individual AtT20 cells under voltage clamp conditions. Coverslips containing cells are placed in a custom acrylic chamber of 0.5 ml volume with a round glass coverslip (25 mm) forming the bottom. The chamber is then transferred to the stage of an inverted microscope (Nikon Diaphot TMD) outfitted for Hoffman modulation contrast and fluorescence optics. Cells are
Introduction of Peptides into the Cell Interior
GoLoco motif peptides are added to the internal pipette solution at a final concentration of 10 μM and diffuse into the cell interior following attainment of the whole cell recording configuration. To first determine whether molecules the size of these peptides (3 to 4 kDa) could diffuse into the cell within the time frame of a typical experiment, we assessed the rate of diffusion of 10 kDa rhodamine dextran B (10 μM, Molecular Probes) from the patch electrode into the cell. In such tests we
Local Application of Agonists to Single Cells
To limit and control application of agonists to the cells of interest during an experiment we employ a planar multibarrel pipette array. The array is constructed from polyimide-coated quartz capillaries (Polymicro Technologies, Phoenix, AZ) attached together with cyanoacrylate glue. We use five to seven capillaries with an inside diameter of 250 μm (Polymicro Technologies) that are cut to the desired length with scissors and filed to a square tip by circular polishing on a fine jeweler's stone.
Data Acquisition and Analysis
Stimulation protocols are generated and data are acquired and digitized using an Axopatch 200B patch clamp in combination with pClamp/Clampex software (v8.2; Axon Instruments, Burlingame, CA). To monitor GIRK currents, a step-ramp protocol consisting of a step from a holding potential of −60 to −100 mV followed by a 0.4-mV/ms ramp from −120 to 40 mV (Fig. 2B) is repeated every 2 to 5 s during changes in external solution. Currents at the end of the step are averaged and plotted to monitor
GoLoco Peptides and GIRK Current Dynamics
Prior to activation of GPCRs by agonist application to a resting cell, the overwhelmingly dominant configuration of the pertussis toxin–sensitive Gi/o protein complement is the heterotrimeric form in which GDP-bound Gα subunits are complexed with Gβ and Gγ subunits. Upon agonist application, the Gα subunit exchanges GDP for GTP and the heterotrimeric complex dissociates. Following hydrolysis of GTP by the intrinsic GTPase activity of the Gα subunit, the free GDP-bound form can reassociate with
GoLoco Peptides and Calcium Channel Dynamics
Several GPCRs couple to the inhibition of calcium channels in neurons (Dolphin 2003, Kuzhikandathil 1999), including D2 receptors, which reversibly inhibit P/Q-type calcium channels in AtT20 cells (Fig. 3A; Kuzhikandathil and Oxford, 1999). In several experiments in which GoLoco peptides were perfused into AtT20 cells expressing D2 receptors, during whole cell recording of barium currents neither R12GL nor AGS3Con peptides altered the degree of inhibition of currents by quinpirole (Fig. 3C,
Conclusions
Investigations of the molecular specificity of G-protein-coupled receptor signaling have employed a variety of approaches during the past decade, including the use of antibodies, antisense knockdown methods, and engineered mutants that are constitutively active, nonfunctional, or dominant negative. Each of these approaches has its advantages and limitations, thus the search for new tools to address these important issues of specificity continues. One of the latest and increasingly popular
Acknowledgements
The authors express their appreciation to Drs. David Siderovski and Randall Kimple for helpful discussions, for initially suggesting the use of GoLoco peptides as probes of GPCR/ion channel coupling, and for providing the peptides used in these experiments. This work was supported by NIH Grants NS18788 to G.S.O. and NS18788-S1 to C.K.W.
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