hDlg is the human homolog of the Drosophila Discslarge tumor suppressor. As a member of the MAGUK (membrane-associated guanylate kinase) family of scaffolding proteins, hDlg is composed of three PDZ (PSD-95, Dlg, and ZO-1) repeats, an SH3 (Src homology 3) motif, and a GUK (guanylate kinase-like) domain. Additionally, hDlg contains two regions of alternative splicing. Here we identify a novel insertion, I1B, located Nterminal to the PDZ repeats. We further analyze the tissue-specific combinations of insertions and correlate those results with the distribution of protein isoforms. We also identify the functions of the two alternatively spliced regions. The N-terminal alternatively spliced region is capable of binding several SH3 domains and also moderates the level of protein oligomerization. Insertions in the second region are responsible for determining the localization of hDlg, with insertion I3 targeting the protein to the membrane regions of cell-cell contact and insertion I2 targeting the protein to the nucleus.First identified from human B lymphocyte cDNA, hDlg encodes a 100-kDa protein sharing ϳ60% similarity with Drosophila Dlg and 70% similarity with rat SAP90/PSD-95 (1). Both hDlg mRNA and protein are abundant in human and murine tissues (1). Consistent with the cellular localization of Dlg to septate junctions, hDlg localizes to regions of cell-cell contact in human epithelial cells (1). Dlg and hDlg are members of the MAGUK 1 family of proteins typically involved in protein scaffolding and cell signaling. MAGUK family members contain three core domains: either one or three PDZ repeats, an SH3 motif, and a GUK domain homologous to guanylate kinase enzymes.hDlg has been shown to interact with a number of cytosolic structural proteins. Protein 4.1 was the first protein shown to bind to hDlg (1). Two members of the ERM (ezrin, radixin, moesin) subfamily of cytoskeletal proteins with homology to protein 4.1 also bind to hDlg and, in turn, form a complex with the membrane glycoprotein CD44 (2). The GUK domain recruits a number of proteins including the GKAP/SAPAP/DAP family of proteins, BEGAIN and MAP1A (3,4).Additionally, hDlg binds to several proteins involved in cell cycle regulation and tumorigenesis. It has been recently demonstrated that PBK, a mitotic serine/threonine kinase, interacts with hDlg (5). The APC tumor suppressor forms a complex with hDlg that blocks progression to the S phase of the cell cycle (6). The human papilloma virus E6 and adenomavirus E4-ORF1 oncoproteins also interact with hDlg. Furthermore, mutant E6 and E4 lacking sequences required for hDlg binding loose their oncogenic capacity (7,8).Several alternatively spliced isoforms of hDlg have been described (Figs. 1 and 6). An alternatively spliced insertion called I1 (1) is located between the unique N-terminal domain of hDlg (NAG) and the first PDZ repeat. The region between the SH3 and GUK domains of hDlg was first characterized as containing two alternatively spliced insertions, I2 and I3 (1). In the same region, a third altern...