KIF1Bβ-Y1087C protein reduced the affinity to IGF1R. (A) An American pedigree of CMT2A1 patients. The proband (Patient 2 in Table 1) was a 43-yr-old male with KIF1Bβ Y1087C mutation genetically confirmed, and so was his 48-yr-old brother. Their mother was also clinically diagnosed as CMT2 and deceased in her 60s, while their father was asymptomatic and healthy at the age of 72 yr. (B) Amino acid sequence alignment of the human KIF1Bβ stalk region spanning the Y1087C mutation between WT and the new CMT2A pedigree (Mut) together with WT mouse KIF1Bβ. (C and D) The GST-pulldown assay of mouse brain lysates using GST-mouse KIF1Bβ 885–1,410 aa with or without the Y1087C mutation immunoblotted with the indicated antibodies (C) and its quantification (D). The signal intensities were normalized by the respective GST-tagged protein expression levels. Note that the binding capacity to IGF1R but not that to DENN/MADD or synaptophysin was significantly decreased by the mutation. CBB, Coomassie Brilliant Blue. n = 3. *, P < 0.05, one-sided paired Welch’s t test. (E–G) Superresolution immunocytochemistry of hippocampal neurons at DIV7 against DENN/MADD (green) and IGF1Rα (red) at low (E) and high (F) magnifications, respectively, followed by the quantification (G). Bars: 10 µm (E); 1 µm (F). Note that there are few colocalizing vesicles with ∼17% for DENN/MADD (n = 142) and 8% for IGF1R (n = 299) of the total vesicles, respectively.