PDGFRα/β heterodimer activation negatively affects downstream ERK1/2 signaling and cellular proliferation.

The platelet-derived growth factor receptor (PDGFR) family of receptor tyrosine kinases allows cells to communicate with one another by binding to growth factors at the plasma membrane and activating intracellular signaling pathways to elicit responses such as migration, proliferation, survival and differentiation. The PDGFR family consists of two receptors, PDGFRα and PDGFRβ, that dimerize to form PDGFRα homodimers, PDGFRα/β heterodimers and PDGFRβ homodimers. Here, we overcame prior technical limitations in visualizing and purifying PDGFRα/β heterodimers by generating a cell line stably expressing C-terminal fusions of PDGFRα and PDGFRβ with bimolecular fluorescence complementation fragments corresponding to the N-terminal and C-terminal regions of the Venus fluorescent protein, respectively.

Cellular Uptake of a Fluorescent Ligand Reveals Ghrelin -Acyltransferase Interacts with Extracellular Peptides and Exhibits Unexpected Localization for a Secretory Pathway Enzyme.

Ghrelin acyltransferase (GOAT) plays a central role in the maturation and activation of the peptide hormone ghrelin, which performs a wide range of endocrinological signaling roles. Using a tight-binding fluorescent ghrelin-derived peptide designed for high selectivity for GOAT over the ghrelin receptor GHSR, we demonstrate that GOAT interacts with extracellular ghrelin and facilitates ligand cell internalization in both transfected cells and prostate cancer cells endogenously expressing GOAT. Coupled with enzyme mutagenesis, ligand uptake studies support the interaction of the putative histidine general base within GOAT with the ghrelin peptide acylation site.

PDGFR dimer-specific activation, trafficking and downstream signaling dynamics.

Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in multiple cellular processes during development. The two PDGFRs, PDGFRα and PDGFRβ, dimerize to form homodimers and/or heterodimers. Here, we overcome previous limitations in studying PDGFR dimer-specific dynamics by generating cell lines stably expressing C-terminal fusions of each PDGFR with bimolecular fluorescence complementation (BiFC) fragments corresponding to the N-terminal or C-terminal regions of the Venus fluorescent protein.

The ghrelin -acyltransferase structure reveals a catalytic channel for transmembrane hormone acylation.

Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound -acyltransferase (MBOAT), ghrelin acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin.