Abstract
The biosynthesis of proteins containing cysteine-rich domains requires chaperones for their correct folding. For instance, the 39-kDa receptor-associated protein (RAP) aides in the cell-surface targeting of newly synthesized members of the mammalian low density lipoprotein receptor (LDLR) gene family, which contains tandemly arranged clusters of hexacysteine repeats. In the chicken, an LDLR relative with eight such repeats is expressed as two different splice variant forms in cell type-specific fashion (Bujo, H., Lindstedt, K. A., Hermann, M., Mola Dalmau, L., Nimpf, J., and Schneider, W. J. (1995) J. Biol. Chem. 270, 23546-23551). To learn more about evolutionary aspects of RAP, its role in escorting of these different receptor splice variants, and other potential functions, we have extended our studies on the avian LDLR family to RAP. cDNA cloning, determination of tissue expression at both the transcript and the protein level, stable expression in COS cells, and binding studies with chicken RAP revealed that mammalian RAPs have retained many features of the non-amniotic proteins. However, structural details, e.g. the well defined internal triplicate repeats in the chicken protein, have been somewhat diluted during evolution. Interestingly, chicken RAP was found to correlate positively with the expression levels in somatic cells of the larger splice variant of the eight-cysteine repeat receptor, but not with those of the smaller variant, expressed only in germ cells. This is compatible with the possibility that RAP may play a role in receptor biology that could be complementing its function in assisting folding. Chicken RAP in crude extracts of the stable expressor COS cells is able to bind to LDLR relatives in ligand blots without requirement for prior purification of the ligand. Thus, in conjunction with the avian model of massive lipid transport to germ cells, these cells provide a novel comparative system amenable to investigation of the biological functions of RAP.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ001912
This notion is based on the known competitive displacement of ligands of the low density lipoprotein receptor (LDLR) family by intracellular receptor-associated protein (RAP), believed to bind to the so-called LDLR binding repeats, which are six cysteine-containing subdomains of ϳ40 amino acids each, clusters of which constitute the ligand binding domains of LDLR family members [40]
RAP plays an important role in receptor folding itself, as shown, e.g. by the reduction of intracellular aggregation of soluble minireceptor forms of LDLR-related protein [8]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ001912. Chickens are not known to harbor an apolipoprotein E gene [24], yet the best studied member of the avian LDLR gene family, the oocyte-specific splice variant of the receptor for very low density lipoprotein and vitellogenin, termed LR8Ϫ [17, 18], has the ability to bind the mammalian apolipoprotein [25].
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