Human Soluble Neuropilin-1(sNRP1) ELISA Kit

Neuropilins (NRPs) are single-pass transmembrane receptors involved in several signaling pathways that regulate key physiological processes such as vascular morphogenesis and axon guidance. The MAM domain of NRP, which has previously been implicated in receptor multimerization, was the only portion of the ectopic domain of the NRPs for which the structure, until now, has been elusive. Using site-directed mutagenesis in the linker region preceding the MAM domain we generated a protein construct amenable to crystallization. Here we present the crystal structure of the MAM domain of human NRP1 at 2.24 angstrom resolution. The protein exhibits a jelly-roll topology, with Ca2+ ions bound at the inter-strand space enhancing the thermostability of the domain. We show that the MAM domain of NRP1 is monomeric in solution and insufficient to drive receptor dimerization, which leads us to propose a different role for this domain in the context of NRP membrane assembly and signaling.

Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF(165)a, VEGF(165)b, and VEGF(121)a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF(165)a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF(165)a > VEGF(189)a > VEGF(145)a. VEGF(165)b, VEGF-Ax, VEGF(121)a, and VEGF(111)a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF(165)a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.