Figure 6.

Identification of the C-terminal interacting domain in PKD2. (A) Direct interaction between the C-terminal tail of PKD2 and TRPC1. In vitro column binding of ³⁵S-PKD2 and ³⁵S-CaM to GST-TRPC1/D⁶³⁹–S⁷⁵⁰ or GST-TRPC1/S⁶⁶²–S⁷⁵⁰ (lanes 1–4). Lane 1 shows 0.1× the input amount of ³⁵S-PKD2 and ³⁵S-CaM. Bound ³⁵S-PKD2 and ³⁵S-CaM to GST, GST-TRPC1/D⁶³⁹-S⁷⁵⁰, or GST-TRPC1/S⁶⁶²–S⁷⁵⁰ is shown in lanes 2–4 and the immobilized amounts of GST, GST-TRPC1/D⁶³⁹–S⁷⁵⁰, and GST-TRPC1/S⁶⁶²–S⁷⁵⁰ subjected to in vitro column binding are shown in lanes 7–9. To obtain an estimate of the immobilized amounts of GST, GST-TRPC1/D⁶³⁹–S⁷⁵⁰, or GST-TRPC1/S⁶⁶²–S⁷⁵⁰ used in the in vitro column binding assays, 1 or 5 μg of purified GST is shown in lanes 5 and 6. A lower molecular weight band corresponding to a C-terminal proteolytic product of ³⁵S-PKD2 is shown by an arrow below the band corresponding to full-length ³⁵S-PKD2. (B) cDNAs corresponding to the entire C-terminal cytoplasmic region of PKD2, systematic N- or C-terminal deletions of this region (amino acid residues 679–968) or nonconserved substitutions in the region 822–895 were tested for their interaction with the cytoplasmic tail of TRPC1 (D⁶³⁹–S⁷⁵⁰) or PKD1 (P⁴¹²⁴–T⁴³⁰³) by the yeast two-hybrid assay. Positive interactions were scored for both survival in plates lacking histidine (his+) and production of β-galactosidase (lacZ+).