Interaction between RGS7 and polycystin (protein–protein interactionyyeast two-hybrid systemypolycystic kidney disease)

Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of certain Ga subunits and thereby modulate a number of G proteindependent signaling cascades. Currently, little is known about the regulation of RGS proteins themselves. We identified a short-lived RGS protein, RGS7, that is rapidly degraded through the proteasome pathway. The degradation of RGS7 is inhibited by interaction with a C-terminal domain of polycystin, the protein encoded by PKD1, a gene involved in autosomal-dominant polycystic kidney disease. Furthermore, membranous expression of C-terminal polycystin relocalized RGS7. Our results indicate that rapid degradation and interaction with integral membrane proteins are potential means of regulating RGS proteins. The recently discovered RGS family consists of negative Regulators of G protein Signaling characterized by an RGS domain of '120 aa (reviewed in refs. 1–5) that allow members to accelerate the intrinsic GTPase activity of Gai and Gaq subunits and thereby terminate downstream G protein-linked signaling pathways (6–14). RGS proteins catalytically interact with their respective Ga subunits; a single molecule binds and inactivates multiple GTP-bound Ga subunits. Thus, tight regulatory mechanisms are required to curtail this enzymatic activity (reviewed in ref. 2). Some RGS proteins are transcriptionally regulated, transiently expressed only after cellular activation (reviewed in refs. 3 and 15). The function of other RGS proteins may depend on subcellular localization (16–18). Several investigators postulate that posttranslational modification of Ga target proteins may modulate the enzymatic activity of RGS proteins. For example, palmitoylation or protein kinase C-dependent phosphorylation of certain Ga subunits decreases their affinities for RGS proteins; these activated Ga subunits are thereby resistant to the GTPase activity of RGS proteins (19, 20). Many regulatory proteins are short-lived intracellular proteins that contain motifs subject to ubiquitin-dependent proteasome degradation (reviewed in ref. 21). A partially conserved 9-aa destruction box is located '40–50 residues from the N terminus of several cell-cycle regulators. Other proteins follow the N-end rule, in which rapid degradation is triggered by basic or bulky-hydrophobic N-terminal amino acid residues (reviewed in ref. 22). The presence of PEST sequences, regions enriched in proline, glutamate, serine, and threonine residues, can also target proteins for degradation (reviewed in ref. 23). We found that RGS7 is a short-lived protein that is rapidly degraded by the ubiquitin–proteasome pathway. It contains two putative PEST sequences that flank a potential coiled-coil domain. Utilizing the yeast two-hybrid system, this region of RGS7 was found to interact with the C-terminal domain of polycystin. PKD1, the gene encoding for polycystin, is mutated in '85% of patients with autosomal-dominant polycystic kidney disease, a common hereditary disease (1:1,000) with progressive epithelial cyst formation that accounts for 8–10% of end-stage renal disease (24). Affected individuals commonly develop kidney and liver cysts and nearly half require renal replacement therapy by age 60. Polycystin is a glycoprotein with multiple transmembrane domains and a Cterminal cytoplasmic tail of 226 aa (25, 26). The N-terminal extracellular region of over 2,500 aa contains several domains with significant homology to membrane proteins involved in cell–cellymatrix interactions. However, the precise function of polycystin remains unknown. We found that the C-terminal domain of polycystin prolonged the half-life of RGS7 and altered its subcellular localization. The regulation of an RGS family member by polycystin may represent a prototypic model of a more prevalent mechanism for regulating G protein signaling by integral membrane proteins. MATERIALS AND METHODS Plasmids. The C-terminal cytoplasmic domain of wild-type PKD1 was amplified by PCR from KG8 (M.C. Schneider, Brigham and Women’s Hospital, Boston). DNA fragments for PKD1 and RGS7 deletion constructs were obtained by PCRdirected introduction of in-frame stop codons. For the yeast two-hybrid analysis, the fragments were cloned into pLEX, a derivative of plex202, to generate a LexA fusion protein, or into pCGA, a derivative of pcgatrp2, containing a bacterial transcriptional activator under the control of the galactoseinducible promoter GAL1 (E.K. and B. Seed, unpublished data). The construct Pkd1.1 contains the C-terminal 226 aa of polycystin from E4078 to T4303, Pkd1.2 contains amino acids E4078–L4266, Pkd1.3 contains E4078–A4232, Pkd1.4 contains S4192–T4303, Pkd1.5 contains R4228–L4266, and Pkd1.6 contains E4078–S4169. The CD16.7 and sIg.7 fusions have been described (27). The Flag-tagged RGS7 constructs contain the Flag-Histag sequence DRKLATMLDYKDDDDKHHHHHHHHH; the complete coding region of RGS7 was fused to the Flag-His tag to yield F.RGS7. RGS7.1 represents L50–Y469; RGS7.2, Q170–Y469; RGS7.3, Q170–R437; RGS7.4, Q170–W330; RGS7.5, Q170–T247; RGS7.6, P253–Y469; RGS7.7, M220–W330; and RGS7.8, E315–Y469. A plasmid directing the expression of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: GGL, Gg-like; RGS, regulator of G protein signaling; HA, hemagglutinin; GST, glutathione S-transferase; GSK-3b, glycogen synthase kinase-3b. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF090116 and AF090117). §To whom reprint requests should be addressed at: Beth Israel Deaconess Medical Center, Renal Division, DANA 517, 330 Brookline Avenue, Boston, MA 02215. e-mail: gwalz@ caregroup.harvard.edu.