UCSF

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that transduce extracellular cues into intracellular signaling responses. Binding of growth factors to the extracellular domains of RTKs promote receptor dimerization and activation of the intracellular kinase domain, resulting in phosphorylation and amplification of downstream signaling pathways. The MET family of RTKs consists of two receptors: the MET and RON receptor. Precise regulation of the MET family of RTKs must be maintained, as aberrant activation of either receptor has been shown to promote tumorigenesis in various cancers. Very little is known about what domains in MET and RON contribute to autoregulation, how binding to their respective growth factors result in an active dimeric complex, and whether additional co-factors or co-receptors contribute to the regulation and stabilization of the active signaling complex. Our studies aimed to close this gap in understanding of MET and RON receptor activation and regulation through structural and biochemical analysis.In Chapter 1, we identify a soluble isoform of the co-receptor Neuropilin-1 (sNRP1) as a previously unrecognized positive regulator of MET signaling. Using cell-based signaling assays, we discovered that sNRP1 enhances MET phosphorylation and downstream signaling. Biochemical analysis revealed that sNRP1 directly binds to MET’s growth factor, HGF, and that HGF mediates the formation of a ternary complex between sNRP1 and MET. AlphaFold 3 structural predictions of the ternary complex suggest that the complex adopts a symmetric architecture in which sNRP1 stabilizes an extended conformation of HGF. Functional validation of this model was performed by mutational analysis of putative interfaces. Collectively, these findings establish sNRP1 as positive modulator of MET signaling where sNRP1 enhances by remodeling the active HGF-MET signaling complex.In Chapter 2, we investigate the molecular mechanism for RON activation and regulation through both its extracellular and intracellular domains. Specifically, we aimed to gain structural understanding of the apo-RON extracellular domain (ECD), its growth factor, MSP, and an active RON/MSP complex. Although challenges existed with the expression and purification of RON ECD which hindered structural studies, these efforts identified key technical barriers and led a foundation for future optimization and purification strategies. In its intracellular region, we were able to identify the juxtamembrane domain of RON as a negative regulator of kinase activity, as its presence decreased the rate of the kinase in-vitro. Together, these findings show a molecular mechanism for autoregulation of RON in its intracellular domain and pave a path towards structurally characterizing the active RON/MSP complex.