Multiple allostery in the regulation of PDGFR beta kinase activities

Abstract

Platelet-derived growth factor receptor beta (PDGFRβ), a type III receptor tyrosine kinase (RTK) with a featured kinase insert, regulates important cellular functions. Dysregulation of PDGFRβ is associated with cardiovascular and fibrosis diseases. Thus, its kinase activity needs to be precisely regulated under physiological conditions. Early studies demonstrated that its kinase is autoinhibited by its juxtamembrane segment and activated by transphosphorylation. However, additional mechanisms are required for the comprehensive regulation of the receptor kinase. Herein, we provide evidence that dimerization of activated kinases, autoinhibition by the kinase insert, and dimerization of inactive kinase, all contribute to the regulation of the receptor kinase. Moreover, we find such multiple allosteric regulation is also conserved in other type III RTKs, including colony stimulating factor 1 receptor (CSF1R). Impaired allosteric regulation of CSF1R is associated with malfunctions of microglia and demyelination of neurons in hereditary diffuse leukoencephalopathy with spheroids (HDLS).

1. Introduction

Platelet-derived growth factor receptor alpha (PDGFRα) and PDGFRβ, together with colony stimulating factor 1 receptor (CSF1R), mast/stem cell growth factor receptor (SCFR or c-Kit), and Fms-like tyrosine kinase 3 (Flt-3) receptors, belong to the type III receptor tyrosine kinase subfamily [1–5]. All of them are composed of 5 extracellular Ig-like domains, a single-pass transmembrane domain (TM), an intracellular juxtamembrane segment (JM), a kinase domain splatted apart by a not-well characterized insert (KI), and a C-terminal tail bearing multiple phosphorylation sites (Figure 1A) [1–5]. These receptors regulate important biological functions, including cell proliferation, differentiation, and migration. Dysregulation of these receptors is associated with cancer, cardiovascular and fibrosis diseases [1–5]. Thus, a clear understanding of the precise regulation of these receptors is essential.

The receptors in this subfamily are regulated by ligand-induced dimerization and subsequent activation through transphosphorylation (Figure 1B) [5–8]. In the absence of ligand, these receptors are monomeric and autoinhibited [5,7–9]. Upon binding to the dimeric ligands, these receptors are assembled into dimers so that two kinases in the dimer could trans-phosphorylate each other and be activated [5,7,8]. Crystallographic studies provided further details for these receptor kinases in autoinhibited and transphosphorylation states [10–13]. In the autoinhibited state, the JM of PDGFRα, FLT3, or c-Kit receptor forms a hairpin winding around the αC helix of the kinase and poses the JM tyrosine-containing segment into the active site of the kinase (Figure 1C) [10,12,13]. As such, the JM blocks the kinase from binding to the substrate [10,12,13]. In the transphosphorylation state of c-Kit kinases, the JM of one kinase is latched onto the C-lobe of another kinase so that the two JM Tyrs from the former kinase can get into the active site of the latter for transphosphorylation (Figure 1D) [11]. Moreover, in this transphosphorylation complex, both kinases are adopted in active conformation [11].

Although these early studies provided a basic framework for understanding the regulation of these receptor kinases, some conceptual gaps regarding the transition and stabilization of different conformational states of the kinase are still missing. For example, it was largely unknown: how is the kinase kept in a precise balance between the autoinhibited state and activated state? In addition, the role of the kinase insert and the potential for higher-order oligomerization in regulating kinase activity remained unexplored. This study addresses these gaps by providing evidence for multiple allosteric mechanisms that collectively fine-tune PDGFRβ kinase activity.