The evolved divergence of γ-secretase-susceptibility of homologous proteins Ngfrb and Nradd in zebrafish

Objective NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by gamma-secretase but there is conflicting data around the susceptibility to gamma-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to gamma-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to gamma-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of gamma-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by gamma-secretase. Either NGFR evolved gamma-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to gamma-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to gamma-secretase.