Human corin isoforms with different cytoplasmic tails that alter cell surface targeting

Abstract

Corin is a cardiac serine protease that activates natriuretic peptides. It consists of an N-terminal cytoplasmic tail, a transmembrane domain, and an extracellular region with a C-terminal trypsin-like protease domain. The transmembrane domain anchors corin on the surface of cardiomyocytes. To date, the function of the corin cytoplasmic tail remains unknown. By examining the difference between human and mouse corin cytoplasmic tails, analyzing their gene sequences, and verifying mRNA expression in hearts, we show that both human and mouse corin genes have alternative exons encoding different cytoplasmic tails. Human corin isoforms E1 and E1a have 45 and 15 amino acids, respectively, in their cytoplasmic tails. In transfected HEK 293 cells and HL-1 cardiomyocytes, corin isoforms E1 and E1a were expressed at similar levels. Compared with isoform E1a, however, isoform E1 was more active in processing natriuretic peptides. By cell surface labeling, glycosidase digestion, Western blotting, and flow cytometry, we found that corin isoform E1 was activated more readily as a result of more efficient cell surface targeting. By mutagenesis, we identified a DDNN motif in the cytoplasmic tail of isoform E1 (which is absent in isoform E1a) that promotes corin surface targeting in both HEK 293 and HL-1 cells. Our data indicate that the sequence in the cytoplasmic tail plays an important role in corin cell surface targeting and zymogen activation.

FIGURE 1.

Alternative exons in the corin gene. A, protein domain structure and different cytoplasmic tails of human (h), mouse (m), and rat (r) corin. Tail, cytoplasmic tail; TM, transmembrane; Fz, Frizzled; LDLR, LDL receptor; SR, scavenger receptor. B, alternative exons encoding N-terminal sequences of human and mouse corin. Protein sequences from different exons are color-coded. The asterisk indicates a stop codon. The diamond indicates Met-30. C and D, RT-PCR analysis of alternatively spliced corin mRNA in human (C) and mouse (D) hearts. Oligonucleotide primers based on sequences of different exons are indicated. Predicted PCR fragments from different exon combinations are shown on the left. Negative controls without mRNA templates and positive controls of GAPDH mRNA are not shown.

FIGURE 2.

Corin isoform expression and activation in HEK 293 and HL-1 cells. A, corin domain structure with alternative cytoplasmic tails. Active residues His (H), Asp (D), and Ser (S) in the protease domain and a C-terminal V5 tag (V) are indicated. The arrow indicates the activation cleavage site at Arg-801. A disulfide bond (S-S) connecting the propeptide and the protease domain is shown. B, corin isoform expression and activation analyzed by Western blotting under reducing and nonreducing conditions with recombinant proteins from transfected HEK 293 and HL-1 cells. Vector-transfected cells and activation cleavage site mutant R801A were used as controls. Corin-p, activated corin protease fragment. C, isoform E1 and E1a expression in HEK 293 cells. D, percentage of the activated protease versus zymogen fragments estimated by densitometry. Data are means ± S.D. from three independent experiments. E, Western analysis of pro-ANP processing by isoforms E1 and E1a. F, estimation of pro-ANP processing by densitometric analysis of Western blots. Data are means ± S.D. from three independent experiments.

FIGURE 3.

Cell surface expression of corin isoforms. A, Western analysis of biotin-labeled cell surface proteins in transfected HEK 293 cells expressing corin isoforms E1 and E1a under reducing (left panel) and nonreducing (middle panel) conditions. As a control, total corin proteins in cell lysates were verified (right panel). GAPDH protein was used as another control for cell surface protein labeling (lower panels). B, Western analysis of biotin-labeled cell surface proteins and total lysates from cells with or without trypsin treatment before lysis. Corin-p, activated corin protease fragment.

FIGURE 4.

Glycosidase digestion and flow cytometric analysis of corin isoform proteins. Recombinant corin isoform E1 and E1a proteins were expressed in HEK 293 cells. Cell lysates (A) and biotin-labeled surface proteins (B) were digested with Endo H or PNGase F. Glycosidase-digested corin proteins were analyzed by Western blotting using an anti-V5 antibody. Corin isoforms on the surface of transiently transfected cells were examined by flow cytometry with an anti-V5 antibody as described under “Experimental Procedures.” C, after background subtraction (control cells), the percentage of cells that were FITC-positive on the surface was calculated and is presented in a bar graph (right). The values are representative of data from three independent experiments.

FIGURE 5.

Analysis of human corin mutants with shortened cytoplasmic tails. A, illustration of N-terminal sequences of corin deletion mutants. Plasmids expressing these mutants were transfected in HEK 293 cells. Fz, Frizzled; TM, transmembrane. T, tail. Corin proteins in total cell lysates (B) and biotin-labeled cell surface proteins (C) were analyzed by Western blotting as described under “Experimental Procedures.” Corin-p, activated corin protease fragment.

FIGURE 6.

Analysis of additional human corin mutants with replaced amino acids. Shown are corin mutants E1 D4a–D4d (A and B), E1 D4e–D4g (C and D), and AANN, DDAA, and AAAA (E and F) and their expression in HEK 293 cells. Corin proteins in cell lysates (left panels in B, D, and F) and biotin-labeled cell surface proteins (right panels in B, D, and F) were analyzed by Western blotting. Corin-p, activated corin protease fragment.