PMC

Binding of A30 to HER3ECD and cellular HER3. (A) Binding of A30 to purified HER3ECD, as determined by the replacement of radiolabeled A30 by unlabeled A30 in a gel-shift analysis, indicates a small number of high-affinity binding sites in the absence of hrg (○). Addition of hrg (•) generates additional binding sites of lower affinity. (B) Binding of A30 to cellular HER3 also shows an increase in binding sites on addition of hrg (•), although less pronounced than in solution. Binding data are shown as displaced labeled A30 (cpm) as a function of unlabeled A30. (C) The Scatchard analysis of data shown in B reveals a smaller increase in high-affinity binding sites parallel with the larger increase in low-affinity sites after addition of hrg (•). C in this analysis represents displaced cpm, and L represents the concentration of unlabeled A30 (nM).

4-Thiouracil-substituted A30 photo-cross-links to ERBB3-ECDs in solution. (A) Sequence and predicted secondary structure of A30 and minimal A30 (mA30) [created using mfold (34)]. (B) Experimentally determined spectrum of the light source used for cross-linking after light passes through a plastic cell culture dish (UV, solid line). The normalized excitation spectrum of the light source is superimposed on the absorption spectrum of A30 (dotted line) and 4-thiouracil-substituted A30 (A30^T, dashed line). (C) A30^T (A^T) cross-links to soluble ECDs of ERBB3 but not ERBB2. Recombinant ECDs were detected by their V5 epitope tags after cross-linking and SDS–PAGE analysis. Cross-linking does not occur for unmodified A30 (A) or with the ECD of ERBB2. Both A30^T (A^T) and minimal A30^T (mA^T) cross-link with high efficiency, and A30 but not mA30 cross-links with low abundance in a 1:2 stoichiometry of ECD to aptamer (indicated with an asterisk). (D) The chemical cross-linking of ERBB3-ECDs in solution is disrupted by binding of A30 to the ECD. The homobifunctional cross-linker BS³ was used in the presence and absence of a 5-fold molar excess of either A30 or NRG1 over ERBB3-ECDs (500 nM). The V5 epitope tag of the ECD was visualized by Western blotting. For NRG1, the disruption of ECD interactions leading to cross-linked dimers (D) coincides with the emergence of a chemically cross-linked NRG1-ECD species (ML) in addition to the ECD monomer (M). BS³ targets primary amines and does not cross-link protein and RNA. (E) A30, NRG1, and ERBB3-ECD form a ternary complex. Biotinylated mA30 (mA30^B) (1 μM), NRG1 (1 μM), and ERBB3-ECD (300 nM) were incubated in the indicated combinations. mA30^B and interacting species were pulled down with agarose-coupled streptavidin and analyzed by SDS–PAGE and Western blotting. The V5 epitope tag on the ERBB3-ECD was visualized, and anti-NRG1 antibody was used to detect NRG1.

A30 and G7 are phosphorylated on serine residues in vivo in an F10-dependent manner; A30 is a substrate for the F10 and B1 kinases in vitro (A) Phosphorylation of the A30 protein in vivo. BSC40 cells were infected with the indicated viruses {wt, ts28 at 39.7°C [tsF10(−)], or vindH1 in the absence of IPTG [H1(−)]} at an MOI of 5 and pulse-labeled with ³²PPi from 6 to 9 hpi; lysates were subjected to immunoprecipitation analysis with the α-A30 serum. The immunoprecipitates and lysates were resolved by SDS-PAGE; the latter were visualized by autoradiography, and the former were subjected to immunoblot analysis with the α-A30 serum (I.B.). Filled and empty triangles mark the differential migration of phosphorylated and unphosphorylated A30 (A30 I.B.) An additional sample of ³²P-A30 was retrieved by immunoprecipitation from wt-infected lysates and subjected to phosphoamino acid analysis (PAA). The positions to which phosphoamino acid markers (P-ser, P-thr, and P-tyr) migrated are indicated by the wickets; the radiolabeled phosphoamino acids derived from A30 were visualized by autoradiography. (B) Phosphorylation ofthe G7 protein in vivo. Phosphorylation of the G7 protein was analyzed exactly as described above for A30 in panel A, except that a polyclonal α-G7 serum was used. (C) Recombinant N′his-A30 is a substrate for both the F10 and B1 kinases in vitro. In the left panel, purified F10 kinase was assayed alone (lane 1), in the presence of the generic substrate MBP (lane 2), or in the presence of recombinant N′his-A30 (lane 3). All reactions were performed at 25°C for 30 min in the presence of [γ-³²P]ATP. Autophosphorylation of F10 (▵), phosphorylation of MBP (◂), and phosphorylation of A30 (•) were readily observed. In the right panel, recombinant B1 kinase was assayed alone (lane 1), in the presence of the generic substrate casein (lane 2), or in the presence of N′his-A30 (lane 6). Autophosphorylation of B1 (▵), phosphorylation of casein (◂), and phosphorylation of A30 (•) were readily observed. The electrophoretic migration of molecular weight markers, with their masses indicated in kilodaltons, is shown at the left of each autoradiograph.

Immunoprecipitation analysis of G7 and A30 expression; A30 and G7 do not coprecipitate, but A30 associates with the F18 protein. (A) Immunoprecipitation analysis of nascent A30, G7, and A14. BSC40 cells infected with wt virus at an MOI of 5 were labeled with [³⁵S]methionine from 6 to 9 hpi. Lysates were subjected to immunoprecipitation analysis with α-A30, α-G7, and α-A14 sera. The precipitates were resolved by SDS-PAGE and visualized by autoradiography. Each serum retrieved the cognate protein; in addition, the α-A30 precipitate contained a strongly labeled species of ∼11 kDa (), and the α-A14 precipitate contained the ∼21-kDa A17 protein, as well as A14 and a small amount of glycosylated A14 (A14 glyco). No interaction between A30 and G7 was detected. The precipitated proteins are identified at the right; molecular markers are shown at the left, with their masses given in kilodaltons. (B) The 11-kDa protein coprecipitating with A30 migrates with A13 and F18 and is not retrieved when F18 expression is repressed. Cells were infected with either wt virus (lanes 1 to 3) or the inducible F18 recombinant in the absence of IPTG (vF18[−], lanes 4 to 6). Cells were metabolically labeled and subjected to immunoprecipitation as described for panel A. (C) The 11-kDa protein that coprecipitates with A30 is the viral F18 protein. Cells infected with wt virus (as described in panel A) were pulse-labeled with either [³⁵S]methionine or [³²P]Pi and subjected to immunoprecipitation with α-A30. The immunoprecipitates were resolved in duplicate by SDS-PAGE; one set was visualized directly by autoradiography (I.P.; top), and the second was subjected to immunoblot analysis with α-F18 serum (I.B.; bottom).