title: Calcium-dependent stability studies of domains 1 and 2 of epithelial cadherin.
abstract: Epithelial cadherin is important in establishing and maintaining cell to cell interactions in epithelial cells, thereby playing an important role during morphogenesis. The epithelial cadherin molecules have three main regions: the N-terminal extracellular region, the transmembrane region that spans the cell membrane once, and the C-terminal cytoplasmic region that communicates with the cytoskeletal actin filaments through catenins. We report studies of the calcium-dependent stability of extracellular domains 1 and 2 of epithelial cadherin as a two-domain construct (MECAD12). Circular dichroism (CD) spectra of MECAD12 indicated a typical beta-sheet conformation in all solution conditions. Thermal- and denaturant-induced unfolding was monitored by CD. Distinct calcium stabilization was observed as a shift in T(m) from 40 (apo) to 65 degrees C (10 mM Ca2+). Spectroscopic experiments agreed well with calorimetric (DSC). In the absence of calcium, the unfolding transition was shallow (deltaH(m) = 40 kcal/mol) but not obviously three state. Model-dependent analysis indicated that a second transition could be assigned to the unfolding of domain 2. A calcium-binding constant was derived from the calcium-dependent shift in temperature denaturation profiles. The Kd that was obtained (55 microM) was consistent with literature values. Thus, the modular domains of epithelial cadherin exhibit context-dependent behavior in both the apo and calcium-bound states. This cooperativity between the modules is consistent with the physiological role of epithelial cadherin in signal transduction through cell-adhesive contacts.
authors.affiliation: Department of Chemistry and Biochemistry, University of Mississippi, University, Mississippi 38677, USA.
authors.forename: Alka
authors.initials: A
authors.lastname: Prasad
authors.forename: Susan
authors.initials: S
authors.lastname: Pedigo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015820:Cadherins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004847:Epithelial Cells / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D015820:Cadherins; D014508:Urea; D002118:Calcium