AGR2 gene function requires a unique endoplasmic reticulum localization motif

Abstract

Soluble proteins are enriched in the endoplasmic reticulum (ER) by retrograde transport from the Golgi that is mediated by the KDEL receptors. In addition to the classic carboxyl-terminal KDEL motif, a variety of sequence variants are also capable of receptor binding that result in ER localization. Although different ER localization signals that exhibit varying affinities for the KDEL receptors exist, whether there are functional implications was unknown. The present study determines whether AGR2 requires a specific ER localization signal to be functionally active. AGR2 is expressed in most human adenocarcinomas and serves a role in promoting growth and the transformed phenotype. Using two different cell lines in which AGR2 induces expression of either the EGFR ligand amphiregulin or the transcription factor CDX2, only the highly conserved wild-type carboxyl-terminal KTEL motif results in the appropriate outcome. Deletion of the KTEL motif results in AGR2 secretion and loss of AGR2 function. AGR2 function is also lost when ER residence is achieved with a carboxyl-terminal KDEL or KSEL instead of a KTEL motif. Thus variations in ER localization sequences may serve a specific functional role, and in the case of AGR2, this role is served specifically by KTEL.

FIGURE 1.

AGR2 expression in rat intestinal IEC-6 cells. A–D, immunofluorescence images of IEC-6 cells transfected with AGR2 constructs terminating with a carboxyl-terminal KTEL (A), KDEL (B), or a STOP inserted before the wild-type KTEL (C) sequence. D, cells transfected with the expression vector alone. A–D, anti-AGR2 antibodies (red) and nuclear DAPI staining (blue). E and F, representative IEC-6 cell transfected with wild-type AGR2 and labeled with anti-KDEL (E, green) and anti-AGR2 (F, red) antibodies. The white bars in the images equal 10 μm. G, real time quantitative PCR determination of AGR2 RNA from transfected IEC-6 cells. The error bars represent S.D. H, protein immunoblots of transfected IEC-6 cell lysates (anti-AGR2 and anti-β-actin) or concentrated media (anti-AGR2). I, Coomassie Blue-stained SDS-PAGE of culture media depicted in H to show protein loading. J, graph of AGR2 protein levels as determined by densitometry of the immunoblot in H. The values are normalized by the corresponding β-actin band density.

FIGURE 2.

AGR2 expression in IEC-6 cells induces CDX2 expression and requires a carboxyl-terminal KTEL sequence. A, real time quantitative PCR determination of AGR2 RNA (gray columns, left ordinate) and CDX2 RNA (black columns, right ordinate) levels in transfected IEC-6 cells. The error bars represent S.D. (n = 2). B, protein immunoblots of IEC-6 cell lysates for CDX2 and β-actin. C–F, immunofluorescence images of IEC-6 cells transfected with the same AGR2 constructs as depicted in Fig. 1A but probed with an anti-CDX2 antibody (red) or DAPI nuclear stain (blue). C, IEC-6:AGR2-KTEL; D, IEC-6:AGR2-KDEL; E, IEC-6:AGR2-STOP; F, IEC-6:VECTOR. The white bars represent 50 μm.

FIGURE 3.

CDX2 induction is mediated by binding to the KDEL receptors. Shown are the effects of expression with GFP and RFP proteins terminated at the carboxyl terminus with KDEL or KTEL on CDX2 expression in IEC-6:AGR2-KTEL cells. A–D show anti-CDX2 (red) and DAPI (blue) staining in IEC-6 cells transfected with AGR2 alone (A), AGR2 + GFP-KDEL (B), AGR2 + GFP-KTEL (C), and expression vector alone and no AGR2 (D). The white bars represent 50 μm. E, proportion of CDX2-positive cells for the constructs shown in A–D as quantified with ImageJ software. The total number of cells counted as determined by DAPI staining is listed in parentheses below the abscissa. F, real time quantitative PCR of CDX2 RNA normalized to β-actin for the same cells shown in E. G, proportion of CDX2-positive cells for similar constructs as shown in E except that RFP was substituted for GFP. H, real time quantitative PCR of CDX2 RNA normalized to β-actin for the same cells shown in G. I–L, controls for GFP transfection efficiency (400× magnification). Phase contrast (I and K) and GFP fluorescence (J and L) of IEC-6-AGR2 cells transfected with GFP-KDEL (I and J) or GFP-KTEL (K and L) are shown. M–P, controls for RFP transfection efficiency. Phase contrast (M and O) and RFP fluorescence (N and P) of IEC-6-AGR2 cells transfected with RFP-KDEL (M and N) or RFP-KTEL (O and P) are shown. Q, culture media of the cells transfected with the GFP constructs were evaluated with protein immunoblotting with anti-AGR2 and anti-KDEL (HSPA5) antibodies. Below each lane is a column graph depicting the density of the bands. R, Coomassie Blue staining of equal amounts of culture media as a loading control.

FIGURE 4.

AGR2 induction of AREG in OE33 esophageal adenocarcinoma cells requires a carboxyl-terminal KTEL sequence. A, real time quantitative PCR of total AGR2 RNA in OE33 cells transiently transfected with AGR2 constructs terminating with a carboxyl-terminal KTEL, KDEL, or a STOP sequence (before the wild-type KTEL). Vector designates transfection with the expression vector alone. B, immunoblots of cell lysates for total AGR2 and β-actin of transfected cells. The AGR2 antisera recognizes both the endogenous and transiently expressed constructs of AGR2. Under each lane is a column graph depicting the total AGR2/β-actin ratio determined by densitometry. C, real time quantitative PCR of total AREG RNA in the same cells as in A. D, determination of AREG protein levels in the culture media by ELISA. E, determination of AREG concentration in the media of wild-type OE33 cells that have been transiently transfected with GFP-KDEL and GFP-KTEL constructs. F–I, controls for GFP transfection efficiency. Phase contrast (F and H) and GFP fluorescence (G and I) of OE33 cells transfected with GFP-KDEL (F and G) or GFP-KTEL (H and I) are shown. The error bars for all of the column graphs represent S.D.

FIGURE 5.

Mutation of KTEL to KSEL results in compromised AGR2 induction of CDX2 and AREG. A and B, immunofluorescence labeling of AGR2-KSEL-expressing IEC-6 cells with anti-AGR2 (A) and anti-KDEL (B) antibodies. The white bars represent 10 μm. C, quantified RNA levels determined by real time quantitative PCR for AGR2 (left ordinate) and CDX2 (right ordinate) for AGR2-KTEL- and AGR2-KSEL-transfected IEC-6 cells. D, proportion of CDX2 positive cells in IEC-6 cells transfected with the AGR2-KTEL or AGR2-KSEL constructs. E and F, representative images from IEC-6 cells transfected with the AGR2-KTEL (E) or AGR2-KSEL (F) constructs and stained for CDX2 (red) and DAPI (blue). The white bars represent 100 μm. G, quantified RNA levels determined by real time quantitative PCR for AGR2 (left ordinate) and AREG (right ordinate) derived from AGR2-KTEL- and AGR2-KSEL-transfected OE33 cells. H, OE33 data from G that depict AREG expression normalized to total AGR2 expression. The error bars on all graphs represents S.D.

FIGURE 6.

IEC-6 and OE33 cells express all three KDEL receptor isoforms. Shown is real time quantitative PCR for the three KDEL receptors (KDELR1, KDELR2, and KDELR3) in human OE33 and rat IEC-6 cells. The ordinate represents RNA levels normalized to β-actin. The error bars represent S.D. of three determinations.

FIGURE 7.

Carboxyl-terminal KDEL and KTEL proteins are both sorted to the same compartment. A–C, confocal imaging of CHO cells transfected with RFP-KDEL (A) and GFP-KTEL (B) in CHO cells. C, merged RFP/GFP image. D–I, IEC-6 cells transfected with AGR2-KTEL (D–F) or AGR2-KDEL (G–I) and probed with anti-AGR2 (D and G) or anti-KDEL (E and H) antibodies. F and I, merged images of the preceding two panels.