A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors

Abstract

Edited by Paul E. FraserThe activity of receptor tyrosine kinases (RTKs) is controlled through their lateral association in the plasma membrane. RTKs are believed to form both homodimers and heterodimers, and the different dimers are believed to play unique roles in cell signaling. However, RTK heterodimers remain poorly characterized, as compared with homodimers, because of limitations in current experimental methods. Here, we develop a FRETbased methodology to assess the thermodynamics of hetero-interactions in the plasma membrane. To demonstrate the utility of the methodology, we use it to study the hetero-interactions between three fibroblast growth factor receptors-FGFR1, FGFR2, and FGFR3-in the absence of ligand. Our results show that all possible FGFR heterodimers form, suggesting that the biological roles of FGFR heterodimers may be as significant as the homodimer roles. We further investigate the effect of two pathogenic point mutations in FGFR3 (A391E and G380R) on heterodimerization. We show that each of these mutations stabilize most of the heterodimers, with the largest effects observed for FGFR3 wild-type/mutant heterodimers. We thus demonstrate that the methodology presented here can yield new knowledge about RTK interactions and can further our understanding of signal transduction across the plasma membrane. Receptor tyrosine kinases (RTKs)2 regulate many key biological processes, including cell survival, growth, differentiation, and migration. There are 58 different RTKs, classified into 20 families based on sequence similarity. An archetypal RTK consists of a ligand-binding extracellular domain, a single-pass transmembrane (TM) domain, and an intracellular (IC) kinase domain (1-5). These receptors are activated upon dimerization, which is known to be a reversible process (6, 7). Dimer formation is required (although not sufficient) for function (2, 6, 8 -11), because it brings the two kinases into close proximity, enabling cross-phosphorylation on specific tyrosines. Phosphorylated RTKs trigger many intracellular signaling cascades, including the MAPK, PI3K, PKC, and STAT pathways. These pathways, in turn, determine cell fate and function (1-5, 12, 13).RTKs play a fundamental role in human development. They are also critical players in the induction and progression of many cancers (1-5, 13-15). Thus, significant efforts have been dedicated to the development of RTK-specific therapies with high specificity and low toxicity. One class of anti-cancer drugs on the market specifically aims to inhibit RTK dimerization, because it is an important regulator of function. The best known example of these drugs is Herceptin, an antibody raised against the extracellular domain of HER2, which is often overexpressed in breast cancer (15, 16). Although Herceptin treatment can significantly improve patient outcomes in some cases, the performance of this treatment and other RTK-targeted molecular therapies has not reached expectations (4,16,17). This may be partly due to gaps in basic knowledge about RTK intera...

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