Human Corin Isoforms with Different Cytoplasmic Tails That Alter Cell Surface Targeting

Xiaofei Qi,Jingjing Jiang,Mingqing Zhu,Qingyu Wu

doi:10.1074/jbc.m110.217570

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.

Highlights

corin zymogen bands (Corin) is a multiple-domain protein, consisting of an N-terminal cytoplasmic tail, a single-span transmembrane domain, and an extracellular region that includes two Frizzled-like domains, eight LDL receptor-like domains, one scavenger receptor-like domain, and one C-terminal protease domain [11, 12]
To examine whether the difference is due to the use of alternative exons, we analyzed genomic sequences of the human and mouse corin genes by BLAST using protein sequences from mouse and human corin cytoplasmic tails
The published human corin N-terminal sequence was from exon 1, whereas that of mouse corin was from exon 1a (Fig. 1B)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—HEK 293 cells were cultured in DMEM with 10% FBS. Murine atrial HL-1 myocytes were provided by William Claycomb (Louisiana State University Medical Center) and cultured in Claycomb medium (JRH Biosciences) with 10% FBS and 4 mmol/liter L-glutamine [19]. All cells were cultured at 37 °C in humidified incubators with 5% CO2 and 95% air. RT-PCR—mRNAs from mouse hearts were used to make cDNAs with the SuperScript III first-strand synthesis system (Invitrogen). PCR to amplify human and mouse corin mRNAs from 5Ј-exons was carried out with primers F1 (5Ј-TGA AAC AGT CTC CTG CCC TC-3Ј), R1 (5Ј-CAT AGG AAA GCA GGA TCA CCA-3Ј), JUNE 10, 2011 VOLUME 286 NUMBER 23

Corin Isoforms and Cell Surface Targeting

RESULTS

DISCUSSION