Functional Analysis of PKD1 Transgenic Lines Reveals a Direct Role for Polycystin-1 in Mediating Cell-Cell Adhesion ANDREW J. STREETS,* LINDA J. NEWBY,* MICHAEL J. O’HARE, † NIKOLAY O. BUKANOV, ‡ OXANA IBRAGHIMOV-BESKROVNAYA, ‡ ALBERT C.M. ONG* *Sheffield Kidney Institute, Division of Clinical Sciences (North), University of Sheffield, Sheffield, UK; † LICR/UCL Breast Cancer Laboratory, University College London, London, UK; ‡ Applied Genomics, Genzyme Corporation, Framingham, Massachusetts. Abstract. The PKD1 protein, polycystin-1, is a large transmem- brane protein of uncertain function and topology. To study the putative functions of polycystin-1, conditionally immortalized kidney cells transgenic for PKD1 were generated and an inter- action between transgenic polycystin-1 and endogenous poly- cystin-2 has been recently demonstrated in these cells. This study provides the first functional evidence that transgenic polycystin-1 directly mediates cell-cell adhesion. In non-per- meabilized cells, polycystin-1 localized to the lateral cell bor- ders with N-terminal antibodies but not with a C-terminal antibody; there was a clear difference in surface intensity between transgenic and non-transgenic cells. Compared with non-transgenic cells, transgenic cells showed a dramatic in- crease in resistance to the disruptive effect of a polycystin-1 antibody raised to the PKD domains of polycystin-1 (IgPKD) in both cell adhesion and cell aggregation assays. The differ- ential effect on cell adhesion between transgenic and non- transgenic cells could be reproduced using recombinant fusion proteins encoding non-overlapping regions of the IgPKD do- mains. In contrast, antibodies raised to other extracellular do- mains of polycystin-1 had no effect on cell adhesion. Finally, the specificity of this finding was confirmed by the lack of effect of IgPKD antibody on cell adhesion in a PKD1 cystic cell line deficient in polycystin-1. These results demonstrate that one of the primary functions of polycystin-1 is to mediate cell-cell adhesion in renal epithelial cells, probably via ho- mophilic or heterophilic interactions of the PKD domains. Disruption of cell-cell adhesion during tubular morphogenesis may be an early initiating event for cyst formation in ADPKD. Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited human renal disease (incidence, 1 in 1000 live births) is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). It is a systemic disorder characterized by the formation of fluid-filled cysts mainly in the kidney but also commonly in the liver and pancreas. ADPKD is also associated with an increased incidence of non-cystic manifes- tations, including hypertension, cardiac valve abnormalities, diverticular disease, and intracranial aneurysms. Since the identification of PKD1 and PKD2, investigation into the putative functions of the two ADPKD proteins, poly- cystin-1 and polycystin-2, has been intense, but a consensus on their likely physiologic functions has not been reached. Several possible mutational mechanisms underlying cyst formation including haploinsufficiency and a two-hit model have been proposed, but it is not clear how mutations in either gene lead to cyst formation (1). Potentially more than one mechanism could be operative. Polycystin-1 is a large (460 kD) heavily glycosylated integral membrane protein (2). It is predicted to have a large N-terminal extracellular domain (approximately 2500 aa), 11 transmembrane domains, and a short C-terminal cytoplasmic tail (3). The extracellular region appears to have a modular structure suggesting the presence of potential functional mo- tifs. These include two leucine-rich repeats (LRR), a C-type lectin, a LDL-A receptor motif and a large region (approxi- mately 1000 aa) with strong homology to the sea urchin re- ceptor for egg jelly (REJ) protein. The major part of the N-terminal region, however, consists of 16 novel repeats (80 to 90 aa) with low sequence homology to Ig domains. These so-called PKD domains or repeats are arranged in tandem (II–XVI) except for domain I, which is present between the LRR and lectin modules. NMR studies of a bacterial recom- binant PKD domain I protein has revealed that it has a -sand- wich fold common to other cell surface proteins (4). The function of the various PKD domains has not been fully elu- cidated. It is notable, however, that synthetic peptides contain- ing a repetitive PKD domain motif (WDFGDG) inhibit ureteric bud branching and nephron formation in organ culture (5) and recombinant PKD domain proteins display strong homophilic interactions in vitro (6). In the latter study, an antibody raised to epitopes within the PKD repeats (IgPKD) as well as recom- Received December 5, 2002. Accepted April 10, 2003. Correspondence to Dr. Albert C.M. Ong, Division of Clinical Sciences (North), University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK. Phone: 44-114-271-4961; Fax: 44-114-256-0458; E-mail: a.ong@sheffield.ac.uk 1046-6673/1407-1804 Journal of the American Society of Nephrology Copyright © 2003 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000076075.49819.9B J Am Soc Nephrol 14: 1804–1815, 2003