Cloning of a novel EGFR-related peptide: a putative negative regulator of EGFR

Abstract

Although epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, differentiation, and transformation in many tissues, little is known about the factor(s) that may modulate its function. We have isolated a cDNA clone from the rat gastroduodenal mucosa whose full length revealed 1,958 bp that contained 227 bp of 5'-untranslated region (UTR) and an open-reading frame encoding 479 amino acids, followed by 290 bp of 3'-UTR. It showed ~85% nucleotide homology to the external domain of the rat EGFR. We refer to the product of the newly isolated cDNA as EGFR-related protein (ERRP). In Northern blot analysis with poly(A)(+) RNA from different rat tissues, ERRP cDNA hybridized to several mRNA transcripts with the strongest reaction noted with a transcript of approximately 2 kb. Maximal expression of the 2-kb mRNA transcript was observed in the small intestine, followed by colon, liver, gastric mucosa, and other tissues. Transfection of ERRP cDNA into a colon cancer cell line, HCT116, resulted in a marked reduction in proliferation in monolayer and colony formation in soft agar compared with the vector-transfected controls. In another colon cancer cell line, Caco-2, with a tetracycline-regulated promoter system, induction of ERRP expression in the absence of doxycycline was associated with a marked reduction in EGFR activation and proliferation. We conclude that the ERRP cDNA may represent a new member of the EGFR gene family and that ERRP plays a role in regulating cell proliferation by modulating the function of EGFR.

Epidermal Growth Factor Receptor–Related Protein Inhibits Cell Growth and Invasion in Pancreatic Cancer

“…Therefore, identification of an inhibitor targeting EGFR is likely to provide a therapeutic benefit for pancreatic cancer. ERRP, a recently isolated pan-erbB inhibitor, possesses substantial homology with the extracellular ligandbinding domain of EGFR and its family members (8). Previously, we reported that ERRP inhibited cell growth of pancreatic cancer cells by attenuating EGFR activation in vitro and in vivo (13,14).…”

“…EGFR-related peptide (ERRP), a recently isolated pan-erbB inhibitor that targets multiple members of the EGFR family, seems to attenuate EGFR activation (8). We have found that recombinant ERRP inhibits the growth of a variety of cancer cells, including colon, prostate, and pancreas in vitro and tumor growth in vivo (9)(10)(11)(12).…”

Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure

“…To develop a cell model for the inducible activation of MLCK, we first established an inducible expression ('Tet-off') system in Caco-2 cells (Yu et al, 2001). These cells were then stably transfected with an inducible construct to express a truncated myosin light chain kinase (tMLCK) lacking the calmodulinbinding inhibitory domain (Ito et al, 1991).…”

“…The Tet-off inducible expression system (Gossen and Bujard, 1992) was established in stably transfected Caco-2BBe cell clones (Peterson and Mooseker, 1992;Yu et al, 2001). These cell lines were then stably cotransfected with a vector derived from pBI-L (Clontech, Palo Alto, CA) that expresses luciferase and truncated myosin light chain kinase (tMLCK) from a single Tet-transactivator transcriptional unit (Baron et al, 1995) and pTK hygromycin (Clontech) and selected using hygromycin.…”

“…Moreover, in the event that MLCK activation alone could trigger TJ regulation, we sought to determine morphological and biochemical changes in TJ structure that accompany MLC phosphorylation-induced barrier function decreases. We developed a Caco-2 cell line stably expressing an inducible transactivating element, Tet-off (Gossen and Bujard, 1992;Yu Journal of Cell Science 119 (10) et al, 2001), that drives expression of a constitutively active truncated mutant of MLC kinase, tMLCK (Guerriero et al, 1986). By suppressing tMLCK expression until the Caco-2 cells formed fully differentiated polarized monolayers, we were able to test the role of MLCK in the acute regulation of TJ function.…”