FIG. 1.

In vitro association of Bα with type I receptors. (A) Association of in vitro-translated Bα with GST-receptor fusion proteins. In vitro-translated ³⁵S-labeled Bα was incubated with glutathione-Sepharose-coupled GST (lane 2) or GST fused to the cytoplasmic domains of Tsk7L/R1 (lanes 3 and 5), TβRI/R4 (lane 4), or TβRII (lane 6). Bound proteins were eluted and separated by SDS-PAGE. Lane 1 shows 1/50 of the input ³⁵S-labeled Bα used in each of the GST adsorption experiments. (B) Lack of interaction of TRIP-1 with the cytoplasmic domain of a type I receptor fused to GST (GST-R1) or with GST itself. (C) Gel electrophoretic analysis of the purified GST fusion experiments used in panel A and other experiments. Whereas the GST protein itself corresponded to a single band on the gel, the fusion proteins ran as several bands, the largest of which corresponded to the full-size proteins, and the smaller bands are presumably degradation products. (D) Covalent cross-linking of Bα in the ABαC complex with the cytoplasmic domain of Tsk7L/R1. Purified ABαC complex was incubated without cross-linking (lane 1) or was chemically cross-linked in the absence (lane 2) or presence (lane 3) of purified cytoplasmic domain of Tsk7L/R1. Cross-linked proteins were separated by SDS-PAGE and immunoblotted with anti-Bα antibody. The arrow points to the cross-linked complex of Bα with Tsk7L/R1 which is detected as an anti-Bα immunoreactive band. The arrowhead points to noncomplexed Bα. (E) Covalent cross-linking of Bβ in the ABβC complex with the cytoplasmic domain of Tsk7L/R1. Purified ABβC was chemically cross-linked in the absence (lane 1) or presence (lane 2) of the His₆-tagged cytoplasmic domain Tsk7L/R1. The arrow points to the cross-linked complex of Bβ with Tsk7L/R1 which is detected as the anti-Bβ immunoreactive band, and the arrowhead points to noncomplexed Bβ. (F) In vitro-translated B′ does not bind GST-receptor fusion proteins. In vitro-translated ³⁵S-labeled Bα (lanes 1 to 3) and B′ (lanes 4 to 6) were tested for their association with GST (lanes 1 and 4) or GST fusion proteins with the cytoplasmic domains of Tsk7L/R1 (lanes 2 and 5) or TβRI/R4 (lanes 3 and 6) as described for panel A.