Cholesterol promotes the formation of dimers and oligomers of the receptor tyrosine kinase ROR1.

ROR1 is a member of the receptor tyrosine kinase (RTK) family that plays a crucial role during organogenesis of bone and neural systems by regulating non-canonical Wnt signaling. Misregulation of ROR1 is additionally a causative factor for carcinogenesis in solid and liquid tumors. However, we have a poor understanding of how ROR1 activity is regulated. We employed a recently developed single-molecule method termed SiMPull-POP to study the oligomeric state of ROR1. RTK function is typically triggered by ligand binding, which promotes self-assembly of RTKs to form dimers and in some cases oligomers. However, our data indicate that ROR1 does not follow this paradigm. Instead, ROR1 forms dimers and oligomers in a process that is not affected by the presence of the ROR1 ligand Wnt5a. Additional experiments indicate that the transmembrane domain of ROR1 has a strong tendency to self-assemble, suggesting that this domain modulates ROR1 dimerization. Investigation into a regulatory mechanism for ROR1 self-assembly led to evaluation of the role of the lipid cholesterol, which plays pleiotropic roles in Wnt signaling. Cholesterol was found to promote the assembly of ROR1, and our results point to the transmembrane domain as the region where cholesterol exerts the regulatory effect. Taken together, our results indicate that ROR1 self-assembles in human cells; however, unlike other RTKs, this process is not stabilized by ligand binding but is instead facilitated by membrane cholesterol.