Purchase this article with an account.
L. Notari, R. Chen, P. Becerra; Structure-Function Relationships of a Pigment Epithelium-Derived Factor (PEDF) Receptor: Defining a PEDF-Binding Region and a Phospholipase A2 Active Site. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2968.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
PEDF is a neurotrophic and antiangiogenic factor for the retina. Recently, we identified a cell-surface receptor for PEDF, PEDF-R, as a transmembrane protein with PEDF binding affinity and a phospholipase A2 activity, activity that PEDF stimulates. In silico analyses of the PEDF-R sequence revealed potential regions for binding PEDF within an extracellular loop L4 (Gly161 - Met325), and putative amino acid positions (Ser47 and Asp166) for a phospholipase A2 active site. The aim of this study is to identify structural determinants for function of the PEDF-R protein.
PEDF-R cDNA with a point mutation (D166A) was prepared. PEDF-R fragments coding for C-end terminal truncations (PEDFR-4, -5, -6, and -7), and for L4 were obtained by PCR amplification using specific primers for human PEDF-R. Bacterial expression vectors were constructed by Gateway technology. The recombinant proteins were synthesized in in-vitro bacterial systems and purified by His-tagged affinity chromatography. Peptides RA and P1-P4 designed from L4 were synthesized. Protein:protein interactions were analyzed by surface plasmon resonance. Phospholipase A2 activity was assayed spectrophotometrically.
Recombinant PEDF-R[D166A] and L4 polypeptides bound to immobilized PEDF in a similar fashion and affinity (KD = 3-8 nM) as wild-type PEDF-R. Recombinant PEDF-R polypeptide fragments with larger truncations from the C-terminal end, PEDFR-4, -5, -6 and -7, bound to PEDF indistinctly. Among the five synthetic peptides, only P1, corresponding to a small central L4 portion, bound to PEDF, but it had lower affinity than wild-type PEDF-R. The truncated polypeptides PEDFR-4, -5, -6 and -7 exhibited phospholipase A2 activity and PEDF stimulated the enzymatic activity of all these proteins as of the wild-type PEDF-R protein. However, the PEDF-R[D166A] and L4 polypeptides lacked phospholipase A2 activity.
These results demonstrate that while the C-terminal region is dispensable for PEDF-binding and phospholipase A2 activity, a small central region within L4 contains structural determinants for PEDF-binding affinity, and amino acid Asp166 is crucial for the enzymatic activity of the PEDF-R polypeptide.
This PDF is available to Subscribers Only