Figure 4.

Mutation of residue R663 did not alter tetramerization of GluR6a receptors at equilibrium. A, Oligomeric states of myc-GluR6a and myc-GluR6a(R663) mutants were analyzed after separation of nondenatured receptor protein on a 3–12% Bis-Tris gradient gel. Proteins were detected with anti-myc antibody in Western blots. Proteins were harvested from COS-7 cells at 48 h after transfection. The receptor proteins were separated into four primary bands with molecular weights predicted to correspond to monomers, dimers, trimers, and tetramers. B, Densitometric line scans of bands in A reveal the density profiles used to measure the migration of the predominant protein bands. T, Tetramer; T′, trimer; D, dimer; M, monomer. C, Native molecular weight markers (dark squares), electrophoresed on the same gel as the receptor proteins, were used to generate a standard curve consisting of a one-phase exponential decay fitted to the data. Migration distances of the predominant bands of the myc-GluR6a receptors were then used to estimate the molecular weights based on the standard curve (empty squares). From the molecular weight of the monomeric subunit, the larger bands were composed of 2.1 subunits (dimer), 3.1 subunits (trimer), and 3.8 subunits (tetramer), confirming our tentative assignment of quaternary structure in A. D, Deglycosylation of myc-GluR6a and R663E receptors with PNGase F caused a shift in molecular weights of monomer and dimer bands to molecular weights similar to those observed in myc-GluR6a(R663A), R663E, and R663Q receptors.