D2 dopamine receptor activation of potassium channels is selectively decoupled by Ga i -specific GoLoco motif peptides Christina K. Webb,* ,1 Christopher R. McCudden, Francis S. Willard, Randall J. Kimple,  David P. Siderovski and Gerry S. Oxford* *Department of Cell and Molecular Physiology and UNC Neuroscience Center and  Department of Pharmacology, Lineberger Comprehensive Cancer Center and UNC Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, USA Abstract The GoLoco motif is a short polypeptide sequence found in G-protein signaling regulators such as regulator of G-protein signaling proteins type 12 and 14 and activator of G-protein signaling protein type 3. A unique property of the GoLoco motifs from these three proteins is their preferential interaction with guanosine diphosphate (GDP)-bound Ga i1 ,Ga i3 and, sometimes, Ga i2 subunits over Ga o subunits. This interaction prevents both spontaneous guanine nucleotide release and reassociation of Ga i -GDP with Gbc. We utilized this property of the GoLoco motif to examine dopamine (D2 and D3) and somatostatin receptor coupling to G-protein-regulated in- wardly rectifying potassium (GIRK) channels in mouse AtT20 cells. GoLoco motif peptides had no effect on either basal channel activity or the initial responses to agonists, suggesting that the GoLoco motif cannot disrupt pre-formed G-protein heterotrimers. GoLoco motif peptides did, however, interfere with human D2 (short) receptor coupling to GIRK channels as demonstrated by the progressively diminished responses after repeated agonist application. This behavior is consistent with some form of compartmentalization of D2 receptors and GIRK channels such that Gbc subunits, freed by local receptor activation and prevented from reforming a heterotrimeric complex, are not functionally constrained within the receptor– channel complex and thus are unable to exert a persistent activating effect. In contrast, GoLoco motif peptides had no effect on either D3 or somatostatin coupling to GIRK chan- nels. Our results suggest that GoLoco motif-based peptides will be useful tools in examining the specificity of G-protein- coupled receptor–effector coupling. Keywords: G-proteins, ion channels, regulator of G-protein signaling proteins, signal specificity. J. Neurochem. (2005) 92, 1408–1418. Neurons and other cells express a large variety of G-protein- coupled receptors (GPCRs) that transduce extracellular signals across the plasma membrane into changes in the activity of a comparably wide assortment of effector enzymes and ion channels (reviewed in Offermanns 2003). Signal transduction between ligand-activated GPCRs and effectors is mediated by membrane-associated heterotrimers composed of Ga,Gb and Gc subunits (Hamm 1998). Ga alternates between a guanosine diphosphate (GDP)-bound inactive state and a guanosine triphosphate (GTP)-bound active conformation through the movement of three flexible ‘switch’ regions (Lambright et al. 1996). The Gbc heterod- imer binds with high affinity only to the inactive conforma- tion of Ga and slows GDP release [acting as a guanine nucleotide dissociation inhibitor (GDI); Higashijima et al. 1987] while facilitating Ga/GPCR coupling. Upon ligand/ receptor interaction, the activated GPCR catalyses release of GDP from Ga, which then binds GTP, releases Gbc and is thus freed to modulate effectors until intrinsic GTPase activity returns Ga to the GDP-bound state. Although originally considered a passive partner of GaÆGDP, the Gbc Received September 17, 2004; revised manuscript received November 8, 2004; accepted November 9, 2004. Address correspondence and reprint requests to Dr Gerry S. Oxford, Stark Neurosciences Research Institute, Indiana University School of Medicine, 950 West Walnut Street, Room 402, Indianapolis, IN 46202, USA. E-mail: goxford@iupui.edu 1 The present address of Christina K. Webb is The Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA. Abbreviations used: AGS, activator of G-protein signaling; D2s, human D2 (short) receptor; GDI, guanine nucleotide dissociation inhibitor; GDP, guanosine diphosphate; GIRK, G-protein-regulated inwardly rec- tifying potassium; GPCR, G-protein-coupled receptor; GTP, guanosine triphosphate; PIP 2 , phosphatidylinositol-(4,5)-bisphosphate; PLC, phos- pholipase C; QPRL, quinpirole; RGS, regulator of G-protein signaling; SST, somatostatin. Journal of Neurochemistry , 2005, 92, 1408–1418 doi:10.1111/j.1471-4159.2004.02997.x 1408 Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 1408–1418