Scale bars: 10 m. We validated the connection of both PLEKHA4-GFP and GFP-PLEKHA4 with endogenous KLHL12 by co-IP followed by westernblot (Number 3B). and PCP signaling pathways through its function as an adaptor that tunes CUL3-KLHL12 activity in the plasma membrane. RESULTS PLEKHA4 Localizes to the Plasma Membrane viaInteractions with PI(4,5)P2 Our desire for PLEKHA4 emerged from a motivation to understand the tasks for phosphoinositides in directing signaling via the engagement of their head group by effector proteins bearing both PH domains and additional domains for mediating signaling. PH domain-containing proteins quantity ~250 in humans, and the majority have not been extensively characterized (Lemmon, 2007). In particular, the PH domain-containing protein PLEKHA4, also known as PEPP1, is portion of a family that includes several mediators of intracellular signaling (e.g., FAPP1/2 [DAngelo et al., 2007; Godi et al., 2004], TAPP1/2 [Li and Marshall, 2015], and PLEKHA7/Hadp1 [Shah et al., 2016]). Other than a single statement suggesting that its PH website binds to phosphatidylinositol 3-phosphate (PI3P) (Dowler et al., 2000) and a computational study predicting that its PH website binds to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) (Jungmichel et al., 2014), PLEKHA4 is an unstudied protein with no known cellular functions. We therefore set out to elucidate its molecular properties, subcellular localization, protein connection partners, and cellular and physiological tasks. We began our studies of PLEKHA4 by analyzing the properties of the PH website and how it influences the subcellular localization of the protein. We found that a fluorescent protein fusion to PLEKHA4 localized to the plasma membrane (Number 1A). This result was surprising because protein-lipid overlay assays experienced previously suggested to other investigators the PH website of PLEKHA4 binds to PI3P, which localizes to endosomes and not to the plasma membrane (Dowler et al., 2000; Schink et al., 2013). Open in a separate window Number 1 PLEKHA4 Localizes to the Plasma Membrane via Lu AE58054 (Idalopirdine) Acknowledgement of PI(4,5)P2(A) Confocal microscopy of HeLa cells transfected with GFP-PLEKHA4. (BCG) Lipid-binding assays via co-sedimentation of PLEKHA4 domains with liposomes. Graphs display the percentage of the protein construct that co-sediments with an excess of liposome of defined composition. (B and C) Co-sedimentation of the wild-type mutants (B) or indicated point mutants (C) of the PLEKHA4 PH website (amino acids 54C167) with liposomes, with 5% of the indicated Lu AE58054 (Idalopirdine) PIP (or 20% of dioleoylphosphatidylserine [PS]) and the remainder as dioleoylphosphatidylcholine (Personal computer) (n = 3). The (C) sign shows no liposomes. (D) Confocal microscopy of HeLa cells transfected having a GFP-tagged PLEKHA4 PH website (GFP-PLEKHA4PH). (ECG) Co-sedimentation of wild-type constructs (E and F) or indicated point mutants (G) of a fusion of amphipathic helix, fundamental peptide, and PH website (PLEKHA4H-BP-PH, amino acids 28C167) with liposomes comprising 5% of the indicated PIP (or 20% PS) and the remainder as Personal computer (E), the indicated concentration of PI (4,5)P2 BMP4 (F), or 5% PI(4,5)P2 (G) (n = 3). (H) Confocal microscopy of wild-type or the indicated mutant of GFP-PLEKHA4H-BP-PH. 4A refers to the quadruple mutant K42A/R43A/R48A/R49A. Level bars: 0 m (A [full size], D, and H); 1 m (A [ inset ]). See also Figure S1. We revisited the PIP binding of the PLEKHA4 PH website (residues Lu AE58054 (Idalopirdine) 45C167) using liposome sedimentation assays that assess protein-lipid relationships in the context of intact lipid bilayers, which represent a more physiologically relevant environment (Zhao and Lappalainen, 2012). The PLEKHA4 PH website partially co-sedimented with liposomes comprising any one of the three bis-phosphorylated PIPs (PI(3,4)P2, PI(3,5)P2, and PI(4,5)P2) and exhibited little affinity for PI3P or the additional PIP varieties (Number 1B). Although moderate, the observed binding Lu AE58054 (Idalopirdine) was specific, as it was abolished from the mutation of either of two important Arg residues in the PH website predicted by a crystal structure to contact the PIP head group (Milburn et al., 2004) (Numbers 1C and S1A). A GFP-tagged PH website used a diffuse cytosolic localization, suggesting that a monomeric PH website was not adequate to confer the membrane focusing on of PLEKHA4 (Number 1D). We noticed that just upstream of the PH website were two additional motifs that could mediate membrane binding: a putative amphipathic helix (H, residues 28C41) and a basic peptide (BP, residues 42C50) (Number S1A)..