Human homologue of the Drosophila discs large tumor suppressor protein forms an oligomer in solution - Identification of the self-association site

The human homologue of the Drosophila discs large tumor suppressor protein (hDlg), a member of the membrane-associated guanylate kinase (MAGUK) superf amily, interacts with K+ channels, N-methyl-D-aspartate receptors, calcium ATPase, adenomatous polyposis coli, and PTEN tumor suppressor proteins, and several viral oncoproteins through its PDZ domains. MAGUKs play pivotal ro les in the clustering and aggregation of receptors, ion channels, and cell adhesion molecules at the synapses. To investigate the physiological basis of hDlg interactions, we examined the self-association state of full-length hDlg as well as defined segments of hDlg expressed as recombinant proteins in bacteria and insect Sf9 cells. Gel permeation chromatography of full-le ngth hDlg revealed that the purified protein migrates as a large particle o f size >440 kDa, Similar measurements of defined domains of hDlg indicated that the anomalous mobility of hDlg originated from its amino-terminal doma in. Ultrastructural analysis of hDlg by low angle rotary shadow electron mi croscopy revealed that the full-length hDlg protein as well as its amino-te rminal domain exhibits a highly flexible irregular shape. Further evaluatio n of the self-association state of hDlg using sedimentation equilibrium cen trifugation,, matrix-assisted laser desorption/ionization mass spectrometry , and chemical crosslinking techniques confirmed that the oligomerization s ite of hDlg is contained within its amino-terminal domain. This unique amin o-terminal domain mediates multimerization of hDlg into dimeric and tetrame ric species in solution, Sedimentation velocity experiments demonstrated th at the oligomerization domain exists as an elongated tetramer in solution, In vitro mutagenesis was used to demonstrate that a single cysteine residue present in the oligomerization domain of hDlg is not required for its self -association. Understanding the oligomerization status of hDlg may help to explicate the mechanism of hDlg association with multimeric K+ channels and dimeric adenomatous polyposis coli tumor suppressor protein. Our findings, therefore, begin to rationalize the role of hDlg in the clustering of memb rane channels and formation of multiprotein complexes necessary for signali ng and cell proliferation pathways.