Revealing Fibroblast Growth Factor Receptor-1 and Klotho-Beta Plasma Membrane Dynamics with FRAP and Number and Brightness Analysis

Abstract

Fibroblast growth factor-21 (FGF21) is a newly appreciated endocrine ligand that signals through fibroblast growth factor receptor-1 (FGFR1). Long term FGF21 treatment has been shown to decrease blood glucose levels in diabetic mouse models. Consequently, the signalling pathway of FGF21/FGFR1 has become a target for the management of diabetes. FGF21 activity requires the expression of a unique co-receptor Klotho-beta (KLB). This dependence suggests FGF21 signals through a FGFR1/KLB complex. To investigate the molecular interactions between FGFR1 and KLB at the plasma membrane, we have created fluorescently-labelled constructs of these receptors. Through FRAP, we revealed that KLB has a significantly shorter recovery half-life than FGFR1. After the addition of lactose, a competitive inhibitor of galectin lattice binding, the recovery half-life of KLB increased with no effect on FGFR1. After addition of FGF21, cells co-expressing KLB and FGFR1 also showed increased recovery half-life. This suggests that KLB interacts with the galectin lattice and co-expression of FGFR1 removes this interaction. To study complex formation of FGFR1 and KLB with the addition of FGF21, we employed the recently developed technique of Number and Brightness Analysis. It was found that KLB oligomerizes greater than 2-fold with the addition of FGF21; however, treatment with lactose abolished this response. In contrast, FGFR1-expressing cells showed no change in oligomerization state. In cells co-expressing KLB and FGFR1, FGF21 induced no significant change in KLB aggregation state while inducing a nearly 2-fold increase in FGFR1. Overall, these data suggest KLB associates with the galectin lattice in the absence of FGFR1, and that FGFR1 and KLB form a 1:2 complex that transitions to 2:2 upon addition of FGF21. Future studies will address how these interactions affect the downstream signalling of this important endocrine factor.