Inhibition of G-protein 13y-subunit functions by phosducin-like protein

Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the fty subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Nati. Acad. Sci. USA 90, 10831-10835]. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PhLP inhibited multiple effects of G-protein Py subunits. First, it inhibited the Pr-subunit-dependent ADP-ribosylation of purified a. by pertussis toxin. Second, it inhibited the GTPase activity of purified G.. The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified P,y subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-Pr-subunit interactions. Therefore, PhLP appears to bind to G-protein Py subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function. Guanine nucleotide binding proteins (G proteins) transmit signals from heptahelical receptors such as rhodopsin or the adrenergic receptors to effectors like adenylyl cyclase and ion channels and thus are key components in the regulation of multiple cellular functions (for reviews, see refs. 1 and 2). They consist of a GTP-binding a subunit and a tightly bound complex of ,B and y subunit, and recent evidence suggests that both the a subunit and the fy-subunit complex can regulate effector molecules (reviewed in refs. 3 and 4). It has long been known that signaling via G-protein-coupled receptors is subject to an array of regulatory mechanisms exerted at the receptor level (5, 6). More recently, however, it has become evident that regulatory mechanisms might also be operative at the G-protein level and that there may be cellular proteins that alter G-protein function and G-protein-mediated signaling. Interactions of such proteins with G proteins may occur via the a or the fry subunits (or both). A class of proteins that has attracted much recent interest are proteins that bind to G-protein fry subunits (Gpy). These include the g-adrenergic receptor kinases (P3ARK), a family of kinases involved in receptor desensitization, phospholipases Co3, and several other cytosolic proteins (7). Many of these proteins contain a structural motif called the pleckstrin homology domain, and it has been shown that the C-terminal half of this motif is essential for the Gy binding of P3ARK and of ras guanine nucleotide releasing factor (7, 8). Phosducin is a cytosolic protein that is most abundantly expressed in the retina and pineal gland (9) but whose mRNA has been found in many other organs (10). Phosducin has high 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. affinity for G-protein fry subunits, and it can inhibit GTPase activity and the signaling function of multiple G proteins (10, 11). Two independent studies have shown that the N terminus of phosducin is critical forG binding (12, 13), and it has been suggested that this region contains the C-terminal half of a pleckstrin homology domain (12). Recently, a cDNA has been isolated from rat brain with high homology to phosducin, and the corresponding protein has been termed phosducin-like protein (PhLP) (14). A 5'-end splice variant of PhLP has been described, termed PhLPL, which contains 79 additional amino acids at the C terminus. Both variants of PhLP show a high degree of similarity to phosducin, but the short N terminus of PhLP is quite different from the much longer one of phosducin (see Fig. 5). Because of the essential role of the N terminus of phosducin for the interaction with G-protein 13Py subunits, the possibility of an interaction of PhLP with G proteins, which was postulated upon its discovery (14), requires experimental examination. In the present study, we have, therefore, expressed and purified PhLP and investigated the interactions of the purified protein with Go protein and its fry subunits. MATERIALS AND METHODS Expression and Purification of PhLP. The cDNA of the short variant of rat PhLP was generated by PCRs with rat brain cDNA as the template and primers corresponding to the 5' and 3' ends of the coding region (plus a 5' restriction site for Nco I or BamHI): 5'-CACCGACGCCA3XGAGCGGCTGATCAAAAA-3' and 5'-GCTGCACGGATCCTCAATCTATTTCTAGATCGCT-3' (start and stop codons are underlined). A 680-bp product was obtained and cloned between the Nco I and BamHI sites of the bacterial expression vector pETlld (Novagen; ref. 15). Automated sequencing confirmed an open reading frame identical to the published PhLP cDNA sequence (14). PhLP was expressed in Escherichia coli strain BL21(DE3) pLysS as described for phosducin (10). Following induction with 0.5 mM isopropyl ,B-D-thiogalactoside (IPTG) for 2 h, the cells were disrupted in a buffer containing 50 mM Tris HCl (pH 7.8), 20 mM EGTA, and 0.1% Triton X-100. DNA was precipitated with 2% streptomycin sulfate followed by centrifugation for 20 min at 50,000 x g. The supernatant was then loaded onto an 8-ml Mono-Q anion exchange column (Pharmacia), which was eluted with a linear gradient of 0-500 mM NaCl in 10 mM Tris HCl (pH 7.4). The eluate was analyzed by SDS/PAGE followed by Coomassie blue staining and by Western blots using an anti-peptide antibody generated in rabbits against the C-terminal sequence CHSEDSDLEID (PhLP 208-218). Peak fractions containing PhLP eluted at -250 mM NaCl. They were pooled, concentrated on Centricon-10 devices to 0.5 ml, and loaded onto a 1.6 x 60 cm Abbreviations: G,y, guanine nucleotide binding protein j3y subunits; IPTG, isopropyl ,B-D-thiogalactoside; PhLP, phosducin-like protein; PARK-1, /3-adrenergic receptor kinase 1. *To whom reprint requests should be addressed.