The endocrine fibroblast growth factor 21 (FGF21) requires both fibroblast growth factor receptor (FGFR) and b-Klotho for signaling. In this study, we sought to understand the inter-molecular physical interactions in the FGF21 ⁄ FGFR ⁄ b-Klotho complex by deleting key regions in FGFR1c or FGF21. Deletion of the D1 and the D1-D2 linker (the D1 ⁄ linker region) from FGFR1c led to b-Klotho-independent receptor activation by FGF21, suggesting that there may be a direct interaction between FGF21 and the D1 ⁄ linker region-deficient FGFR1c. Consistent with this, the extracellular portion of FGFR1c lacking the D1 ⁄ linker region blocked FGF21 action in a reporter assay, presumably by binding to and sequestering FGF21 from acting on cell surface receptor complex. In addition, the D1 ⁄ linker region-deficient FGFR1c had enhanced interaction with b-Klotho. Further, we demonstrated that deletion of the D1 ⁄ linker region enhanced the formation of the FGF21 ⁄ b-Klotho ⁄ FGFR1c ternary complex in both Biacore and asymmetrical flow field flow fraction-ation studies. Finally, we found that the N-terminus of FGF21 is involved in the interaction with FGFR1c and FGF21 ⁄ b-Klotho ⁄ FGFR1c ternary complex formation. Taken together, our data suggest that the D1 ⁄ linker region regulates both the FGF21 ⁄ FGFR1c and FGFR1c ⁄ b-Klotho interaction, and a direct interaction of FGF21 with FGFR1c may be an important step in receptor-mediated FGF21 signaling. Key words: D1 and the D1-D2 linker, FGF21, FGFR1c, b-Klotho Abbreviations: AF4, asymmetrical flow field flow fractionation; D1, immunoglobin domain 1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; HSPG, heparin sulfate proteoglycan; mIMCD3, murine renal inner medullary collecting duct; the D1 ⁄ linker region, D1 and the D1-D2 linker. Fibroblast growth factor 21 (FGF21) and its two other subfamily members, FGF19 and FGF23, have emerged as potent endocrine hormones involved in the regulation of diverse physiological functions (1-3). In particular, FGF21 has generated a lot of excitement for its potential of becoming a therapeutic for type 2 diabetes and dyslipi-demia (4). Noticeably, administration of recombinant FGF21 lowered plasma glucose, insulin, triglycerides, and cholesterol levels and improved insulin sensitivity, hepatic steatosis, and obesity in a range of diabetes ⁄ obesity animal models (3,5-7). The key differences that distinguish the FGF19 subfamily members from paracrine FGFs are their lack of or weak heparin-binding properties (8) and their dependence on Klotho family transmembrane proteins as co-receptor for the activation of FGF receptor (FGFR) sig-naling (9). a-Klotho is required for FGF23 to regulate renal phosphate metabolism (10), whereas b-Klotho is required for FGF21 to regulate glucose and lipid metabolism (11-14) and FGF19 to regulate bile acid metabolism (15,16). Interestingly, FGF19 can also induce signaling with a-Klotho as a co-receptor, (16) although the physiological implication is still unclear. In contrast, paracrine FGFs signal...