Binding affinity of full-length and extracellular domains of recombinant human (pro)renin receptor to human renin when expressed in the fat body and hemolymph of silkworm larvae
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Abstract
Transmembrane domains of some receptors have been found to be very important in the
process of constitutive oligomerization, and in the stability and functioning of the
receptor. In this study, full-length of human (pro)renin receptor (hPRR) and hPRR
lacking cytoplasmic domain (hPRR-DeltaCD) were expressed in fat body of silkworm larvae,
and the extracellular domain of hPRR (hPRR-DeltaTMDeltaCD) in hemolymph. Three forms
of hPRR were used for investigation of the interaction between receptor and ligand
using surface plasmon resonance (SPR). As a result, the cytoplasmic domain was not
an essential requirement for binding affinity, but the transmembrane domain of hPRR
was indispensable in the formation of functional hPRR. The dissociation equilibrium
constants (K(D)) of purified hPRR and hPRR-DeltaCD were estimated to be 46 nM and
330 nM, respectively. No evidence of binding by the extracellular domain of hPRR located
in hemolymph was found. However, the solubilized microsomal fraction of the extracellular
domain of hPRR expressed in the fat body showed specific affinity, but lost its binding
affinity after purification. Its binding affinity was recovered by mixing microsomal
fraction of mock-injected fat body to the purified extracellular domain. It is probable
that an artificial transmembrane domain stabilizes the extracellular domain of hPRR
and native conformation may be structurally recovered. To our knowledge, these are
the first findings describing the interaction of transmembrane and extracellular domains
of hPRR with ligand and this may help towards the understanding of binding affinity
of hPRR to ligand.