Polarized expression of functional rat liver asialoglycoprotein receptor in transfected Madin-Darby canine kidney cells.

L Graeve,A Patzak,K Drickamer,E Rodriguez-Boulan

doi:10.1016/s0021-9258(19)40180-4

Abstract

The rat liver asialoglycoprotein receptor or rat hepatic lectin (RHL) consists of two polypeptide species, a major one designated RHL-1 and a minor one designated RHL-2/3, which exists in two differentially glycosylated forms. We have studied the biosynthesis, targeting, and function of the different forms after transfection of their cDNAs into the polarized Madin-Darby canine kidney cell line. In cells expressing only RHL-1, newly synthesized protein undergoes rapid intracellular degradation and is not detected at the cell surface. In contrast, RHL-2/3 when transfected alone is much more stable and is expressed at the basolateral surface of fiber-grown cells. When both forms are expressed together, newly synthesized RHL-1 escapes rapid degradation and is detected at the basolateral surface. In double transfectants a functional receptor is formed that specifically endocytoses and degrades ligand at the basolateral side.

Highlights

From the Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021 ana’ the
Constructs containing full length cDNAs for rat hepatic lectin (RHL)-1 and RHL-2/3 under the control of a retroviral promoter were transfected into MDCK cells along with pMV6 tk neo (Maddon et al, 1985), a plasmid conferring resistance to the antibiotic G418
Stability of RHL-2/3 was similar in single and double transfectants. These results suggest that coexpression of both forms “rescues” RHL-1 at least partially from rapid intracellular degradation, and that it alters the processing of the precursor RHL-2’ to give rise to two final forms as in the hepatocyte

Summary

PROCEDURES

Celki and Materials-MDCK cells, passage, were grown in Dulbecco’s (DME) (GIBCO) supplemented with. Nitrocellulose sheets were blocked with 2% (w/v) non-fat dry milk (Carnation) in PBS containing 0.5% (v/v) Tween 20 and incubated with RHL-1 or RHL-2/3 antiserum (1:lOOO) followed by ““I-protein A (1 X 10’ cpm) in the same buffer. MDCK monolayers grown in polycarbonate labeled for 20 h with Tra#S-label filter-chambers for 6 days were (150 &i/ml) in DME containing. Monolayers were washed twice for 5 min with 100 pg/ml trypsin-inhibitor added to both sides of the filter and several times with PBS. MDCK monolayers on filter chambers were washed four times with PBS/CM for 15 min at 4 “C each. Filter grown cells (2.4 x 10” cells/Transwell) were preincubated for 1 h in binding medium at 37 “C, after which radiolabeled ligand was added to either the apical or basolateral compartment. Filters were washed five times with PBS containing 4 mM EDTA in the cold, cut from the filter chambers, and counted

RESULTS

R c a3

DISCUSSION