The N-terminal 81-aa fragment is critical for UT-A1 urea transporter bioactivity

Abstract

The serine protease, furin, is involved in the activation of a number of proteins most notably epithelial sodium channels (ENaC). The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. UT-A1's amino acid sequence has a consensus furin cleavage site (RSKR) in the N-terminal region. Despite the putative cleavage site, we find that UT-A1, either from the cytosolic or cell surface pool, is not cleaved by furin in CHO cells. This result was further confirmed by an inability of furin to cleave in vitro an (35)S-labeled UT-A1 or the 126 N-terminal UT-A1 fragment. Functionally, mutation of the furin site (R78A, R81A) does not affect UT-A1 urea transport activity. However, deletion of the 81-aa N-terminal portion does not affect UT-A1 cell surface trafficking, but seriously impair UT-A1 urea transport activity. Our results indicate that UT-A1 maturation and activation does not require furin-dependent cleavage. The N-terminal 81-aa fragment is required for proper UT-A1 urea transport activity, but its effect is not through changing UT-A1 membrane trafficking.

Fig. 1

A) A consensus furin cleavage site is conserved in rat, human, dog, monkey, but not in cattle. B, C) UT-A1 expression in furin manipulated CHO cells. pEGFP-UT-A1 was transiently transfected into wild type CHO, furin-deficient CHO, and furin-deficient + furin CHO cells for 48 hr. Total cell lysates or cell surface biotinylated proteins were analyzed by immunoblotting with anti-GFP (N-term, B) or anti-UT-A1 (C-term, C) antibodies.

Fig. 2. In vitro

furin cleavage. The whole UT-A1 (A) or a 126 N-terminal fragment (B) was prepared from rabbit reticulocyte lysates and radioactively labeled with 35S-methionine. The recombinant purified products were then processed for enzyme digestion with 1 and 5 units of furin at 30°C for 2 or 16 h. The samples were run on 4-20% SDS-PAGE and detected by autoradiograph. Arrows indicate the cleaved products.

Fig. 3

Disruption of UT-A1 furin sites does not affect UT-A1 activity. Oocytes were injected with cRNAs encoding UT-A1, or UT-A1 mutants (R78A, R81A) for 3 days. Top) urea transport activity was measured by 14C urea flux (n=5∼6 oocytes). Bottom) UT-A1 protein was analyzed by immunoblotting with antibody to UT-A1. Asterisk indicates a non-specific band. The same membrane were striped and re-probed with actin.

Fig. 4

Deletion of N-terminal 81-aa impairs UT-A1 activity. Oocytes were injected with cRNAs of UT-A1 or truncated UT-A1 (Δ81A1) for 3 days (top). Middle) urea transport activity was measured by 14C urea flux (n=5∼6 oocytes). Bottom) Total and biotinylated UT-A1 were analyzed by immunoblotting with antibody to UT-A1. The same membranes were re-probed for actin or detected by ABC (avidin-biotin complex).