Figure 7.

Specific interactions of SGs and DGs. The predicted hamster α-, β-, γ-, and δ-SG polypeptides comprised 387, 320, 291, and 289 amino acids, respectively, and exhibited the following essentially similar structures: (i) a large extracellular domain with a putative N-glycosylation site(s), (ii) a single transmembrane domain, and (iii) a relatively short intracellular domain. The N-terminal domain of α-SG is predicted to be extracellular, due to the presence of a signal sequence. The C-terminal domains of the β-, γ-, and δ-SGs are assumed to be extracellular. α-DG is an extracellular globular glycoprotein and β-DG is another membrane protein with a topography similar to α-SG (14). Accordingly, putative extracellular domains of α-, β-, γ-, and δ-SGs and β-DG are designated as α-SG N, β-SG C, γ-SG C, δ-SG C, and β-DG N. (a) In vitro pull-down study for SGs and DGs. Specific interaction of labeled (input indicated at the bottom) and immobilized (shown in the lanes) proteins is detected as a band. For SGs, reciprocal pull-down combinations were obtained as follows. α-SG-N bound β-SG-C and δ-SG-C. β-SG-C bound α-SG-N, γ-SG-C, and δ-SG-C. γ-SG-C bound β-SG-C and δ-SG-C. δ-SG-C bound α-SG-N, β-SG-C, and γ-SG-C. Strong signals indicate high affinities between the two proteins tested (such as β-SG-C vs. δ-SG-C). None of the intracellular domains of the four SGs and β-DG bound each other (data not shown). α-DG bound α-SG-N, β-SG-C, δ-SG-C, and β-DG-N. β-DG-N bound α-SG-N, β-SG-C, γ-SG-C, and δ-SG-C. In ×0.1 indicates 10% of the input. (b) Ligand overlay assay for DGs. Specific interaction of labeled (incubated; indicated at the bottom) and electrophoresed (shown in the lanes) proteins is detected as a band. CBB, Coomassie staining. The same binding profiles as in a were confirmed.