Fig. 1.

Dimerization of the GLP-1R is important for high-affinity agonist binding and receptor coupling to cAMP production. Saturation (A) and static (B) BRET data from transient coexpression, in Cos-1 cells, of Rluc- and YFP-tagged GLP-1R constructs of wild-type, in the presence or absence of TM4 peptides from the GLP-1R or from the secretin receptor, or dimer disrupting mutants of TM4. In the presence of the GLP-1R TM4 peptide or when TM4 double/triple mutants were present, the static BRET ratio was reduced and was not significantly different from background signal. (C) GLP-1(7-36)NH₂ stimulated cAMP responses in CHO cells stably expressing the GLP-1R in the presence or absence of the GLP-1 receptor TM4 segment peptide. (D) GLP-1(7-36)NH₂ stimulated cAMP responses in Cos-1 cells transiently expressing either the wild-type or L256A, V259A, or G252A, L256A, V259A TM4 mutant GLP-1Rs. The cell surface expression of the G252A, L256A, V259A mutant was 112 ± 10% of the wild-type receptor. (E) Inhibition of ¹²⁵I-GLP-1(7-36)NH₂ binding, to membranes from Cos-1 cells transiently transfected with the wild-type GLP-1R, by unlabeled GLP-1(7-36)NH₂ in the presence or absence of 10 µM GppNHp. (F) Inhibition of ¹²⁵I-GLP-1(7-36)NH₂ by unlabeled GLP-1(7-36)NH₂ in the presence or absence of 10 µM GppNHp, to membranes from Cos-1 cells transiently transfected with the G252A, L256A, V259A mutant GLP-1R. For reference, the curve for the WT receptor is shown with a dashed line. Values are means ± SEM of data from four to five independent experiments performed in duplicate.