The murine haemopexin receptor. Evidence that the haemopexin-binding site resides on a 20 kDa subunit and that receptor recycling is regulated by protein kinase C.
Haemopexin receptors from mouse hepatoma (Hepa) cells were affinity-labelled by cross-linking to haem-125I-haemopexin complexes using two homo-[disuccinimidyl suberate (DSS) and 3,3'-dithiobis(succinimidyl propionate) (DTSSP)] and one hetero-[sulphosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulpho-SMPB)] bifunctional cross-linking agents. Analysis of the cross-linked products by SDS/PAGE in the absence of reducing agents revealed that 125I-haemopexin was cross-linked specifically to a protein of apparent molecular mass 85-90 kDa. Upon reduction, haemopexin remained cross-linked to a protein of 20 kDa, suggesting that the murine haemopexin receptor has a subunit structure. Two subunits were identified: alpha (p65) and beta (p20). Furthermore, because haemopexin was cross-linked by all three agents to p20, the shortest cross-linker arm being 1.1 nm (11 A), we propose that the haem-haemopexin-binding site resides on this subunit. In addition, a cysteine residue of p20 is located near the haemopexin-binding site, since haemopexin, which has no free thiol groups, is cross-linked to this subunit by the hetero-bifunctional agent sulpho-SMPB. Exposure of Hepa cells to the tumour-promoting phorbol ester 4 alpha-phorbol 12-myristate 13-acetate (PMA) causes a rapid redistribution of haemopexin receptors from the cell surface to the cell interior. Within 2-4 min of incubation with 100 nM-PMA, there was an approx. 50% decrease in cell-surface haemopexin receptors, as judged by ligand binding at 0 degrees C and affinity labelling of the receptor. This time- and dose-dependent down-regulation was fully reversible within 60-90 min after removal of PMA, and the affinity of the remaining receptors was unaltered by PMA. The specificity of PMA was demonstrated by comparison with the non-tumour-promoter 4 alpha-phorbol, which did not affect any of the parameters examined. The amine H-7, a specific inhibitor of protein kinase C, antagonised the receptor redistribution effect of PMA, suggesting that the down-regulation of haemopexin receptors on the cell surface was a consequence of protein kinase C activation. The PMA-induced decrease in surface haemopexin receptors was due to a 2-fold increase in the rate of internalization (from 0.73 min-1 to 1.32 min-1), whereas the rate of exocytosis (0.6 min-1) was unchanged. PMA treatment, like binding of the natural ligand, haem-haemopexin, results in a lower steady-state level of surface haemopexin receptors independent of receptor synthesis, and the receptors were not degraded but were recycled back to the cell surface.
Smith
A
A
Department of Biochemistry and Molecular Biology, LSU Medical Center, New Orleans 70112.
Farooqui
S M
SM
Morgan
W T
WT
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015536:Down-Regulation; D004705:Endocytosis / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects; D006466:Hemopexin / Q000378:metabolism*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D046911:Macromolecular Substances; D051379:Mice; D008961:Models, Structural; D008970:Molecular Weight; D011493:Protein Kinase C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D018000:Receptors, Peptide; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C047453:hemopexin receptor; D006466:Hemopexin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate