Fig 6

Fig 6

(A) HEK293 cells expressing GRK2 and either SMO674 (lanes 1–4), SMO674Ala, which carries mutations in 7 GRK2/3 phosphorylation sites (lanes 5–8), or SMO566 (lanes 9–12). Following treatment with SMO modulators or Cmpd101 (4 hours), SMO was isolated via FLAG affinity chromatography, and total protein or phosphoprotein was visualized using Stain-Free imaging or Pro-Q Diamond staining, respectively. Although GRKs often phosphorylate GPCRs on the intracellular loops of their 7TM domains [67,68], we did not observe phosphorylation within this region of SMO via phosphoprotein staining (A) or MS (S10 Fig). Molecular masses are in kDa. (B) Clusters of phosphorylated residues identified by MS are labeled above the sequence of mouse SMO. Orange indicates phosphorylation that depends on SMO and GRK2/3 activity, while yellow indicates non-GRK phosphorylation sites. Alignment with SMO from other species reveals sequence conservation (blue), particularly among vertebrates. Green indicates GRK phosphorylation sites previously mapped in Drosophila Smo [80]. Vertical lines indicate breaks in sequence. See S9A Fig for complete alignment. (C) Targeted MS-based quantification of phosphorylation at each of the 3 activity- and GRK2/3-dependent clusters in the SMO pCT (left 3 graphs) and total SMO protein in each sample (right-most graph). “Intensity” is a measurement of the abundance of phosphorylation sites (left) or total protein (right), derived from model-based estimation in MSstats which combines individual peptide intensities (see “Methods”). (D) BRET between PKA-C and wild-type SMO657 or SMO657Ala. Data in (C): n = 3 biological and 3 technical replicates per condition. Data in (D): n = 3–6 biological replicates per condition. Error bars = SEM. The underlying data for this figure can be found under S6 Data. The uncropped protein gels are included in S8 Data. See S1 Table for statistical analysis. Cmpd101, Compound 101; GPCR, G protein–coupled receptor; GRK, GPCR kinase; KAADcyc, KAAD-cyclopamine; MS, mass spectrometry; pCT, proximal segment of the cytoplasmic tail; PKA-C, PKA catalytic subunits; SMO, Smoothened.