Proteolytic cleavage of polycystin-1. (A) Multiple sequence alignment of GPS domains using clustal. The cleavage site is marked by an arrow. Residue numbers are shown after the name of each protein and the species from which it is derived. Bov, bovine; Ce, Caenorhabditis elegans; Fugu, Fugu rubripes; Spu, Strongylocentrotus purpuratus; LPH1, Latrophilin/CL-1. (B) A schematic diagram of the domain organization of polycystin-1. SP, signal peptide; R1 and R2–16, PKD repeats; CLD, C-type lectin domain. The predicted cleavage site with the adjacent sequence is presented at right. A black bar indicates epitopes recognized by the antibodies used in this study. The positions of Flag-epitope and the GFP-tag are indicated. WT encodes FL polycystin-1 with a Flag-epitope, whereas GFP-WT has both a GFP-tag and a Flag-tag. (C) Western blots demonstrating cleavage of exogenously expressed polycystin-1. HEK cells were either untransfected or transfected with WT or GFP-WT. F6 and C8/68 are MDCK clones with stable expression of either empty vector or human PKD1 (5). The antibodies used for IP and Western blot are indicated. (D) Western blots demonstrating cleavage and tethering of endogenous polycystin-1 in normal human kidney (NHK), human primary umbilical vein endothelial cells (HUVEC), and human primary aortic endothelial cells (HAEC). Lanes 5–8 show the reciprocal co-IP of NTF and CTF using GFP-WT. Nonspecific trapping of the products by either M2 beads or α-GFP was excluded by using an irrelevant antibody for the IP (α-myc). A myc-tagged form of PKD1 (Myc-WT) served as a positive control for the α-myc IP. (E) Autoradiograph of α-Flag IP product from WT-transfected HEK cells that were metabolically labeled for 20 min and then “chased” for various times, as indicated on top. The position of FL and NTF was confirmed on the Western blot by α-LRR (not shown). (F) Mutations in the GPS of polycystin-1. Only residues that differ from the WT polycystin-1 are shown. (G) Effect of amino acid substitutions on cleavage. Each construct carrying the mutation as indicated on top was transfected into HEK cells and assayed for cleavage as shown. E3020X (19) served as a negative control for IP because it lacks the Flag-tag.

Characterization of NTF tethering. (A) Tethering of NTF by CTF. (Left) E3020X was transfected either alone or cotransfected with a CTF construct that includes a C-terminal Flag sequence and assayed for cleavage as shown. Equal amounts of NTF were present in samples with and without CTF coexpression (Lysate). Nonspecific trapping was excluded by using an irrelevant antibody (α-Myc) for IP. (Right) The immunofluorescence results of HEK cells transfected with GFP-E3020X alone or cotransfected with CTF both intracellular and plasma-membrane. (B) Chemical nature of NTF tethering. Co-IP of NTF by CTF-specific α-Flag from WT-transfected HEK cells was tested under native (N), denaturing and nonreducing (D), or denaturing and reducing (D+R) conditions, as outlined (Right). (C) Concentrated culture medium of cells transfected with each of the constructs was analyzed by Western blotting by using α-LRR (Upper). WT-Flag-IP served as size control for FL and NTF. (Lower) Flag-IP for the corresponding protein lysate probed with α-CT. The intracellular level of E3020X was similar to that of WT when total protein lysates were assayed on Western blot by α-LRR (not shown).

Effect of PKD1-associated mutations on cleavage. (A) Schematic structures of five disease-associated mutant forms of polycystin-1 and three normal variants. An arrowhead marks the relative position of the substitution in each protein. “MUT” identifies disease-associated mutations and “POLY” identifies polymorphic variants. (B) The cleavage property of each mutation was analyzed by using either α-CT (Upper) or α-LRR (Lower). The asterisk indicates an additional band of uncertain nature. (C) Cleavage-deficient forms of polycystin-1 traffic normally. GFP-WT, GFP-L2993P, and GFP-Q3016R were expressed in HEK cells and evaluated for the subcellular localization of each. (D) A cell surface biotinylation study was performed on HEK cells transfected with various constructs as indicated. Biotinylated surface proteins were captured by streptavidin beads and analyzed on Western blot using α-CT. WT/Flag-IP without biotin treatment (lane 1) served as control for FL and CTF. E3020X served as negative control for α-CT. Asterisks indicate nonspecific bands of unknown identity.

Effect of cleavage-disrupting mutations on functional properties of polycystin-1. (A) Various forms of polycystin-1 were expressed in MDCK cells by using an episomal expression system and detected by α-LRR antibody on Western blot. E3020X was expressed and loaded in parallel to identify the position of NTF. (B) Quantification of the types of structures formed by MDCK cells expressing various PKD1 constructs when grown in collagen. The parental cell line and cells transfected with empty vector served as negative controls. The total number of structures examined for each experiment is shown at the bottom. (C) Representative examples of the types of structures formed by MDCK cells described in B. (Bars = 100 μm.) (D) Transcriptional activity of STAT1 was measured in HEK cells by using a luciferase reporter assay. The various constructs were cotransfected with pTK-IRF1-Luc and pCMV β-gal. Luciferase activity was measured 24 h after transfection, and activity was normalized to the level of β-gal expression. The results of a single experiment performed in triplicate are presented.