Receptor-associated Protein in an Oviparous Species Is Correlated with the Expression of a Receptor Variant
Journal of Biological Chemistry
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
... 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 nonamniotic 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. Low density lipoprotein receptor (LDLR) 1 gene family mem-bers are characterized by the presence in their extracellular domains of clusters of tandemly arranged repeats, each containing six cysteines. On the cell surface, these clusters of repeats constitute the binding sites for circulating ligands; intracellularly, they are now known to be the target of a 39-kDa protein (1, 2). This protein has been identified in mammals by reproducible copurification with LDLR-related protein, a large member of the LDLR family (3-6), and hence was termed receptor-associated protein (RAP) (5). RAP is a resident protein of the endoplasmic reticulum (ER) that has been implied as chaperone of LDLR gene family proteins, which are characterized by extracellular cysteine-rich repeats (1, 7-9). Due to the high affinity and apparently specific interaction with the ligand binding domains of LDLR family members, RAP has proven useful in studies on the binding properties of receptors in vivo (9, 10), on the surface of cultured cells (11, 12) , and in solid-phase assays (6, 13, 14) . Among the possible physiological ligands of mammalian LDLR family members, apolipoprotein E has particularly high affinity for certain of the receptors. It has been shown that apolipoprotein E, when overexpressed in heterologous cells, leads to aggregation of nascent receptors and that RAP can alleviate such aggregation by intracellular binding to the cysteine-rich domains (15). We are particularly interested in the LDLR gene family of the chicken (16 -20) as well as in the molecular genetics of receptor-mediated lipoprotein metabolism of oviparous species (21-23). 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). LR8 exists in two splice variants, one containing (LR8ϩ) and one lacking (LR8Ϫ) a domain with potential for multiple O-glycosylation (the socalled "O-linked sugar domain" of the LDLR gene family) (18). These different receptor forms are expressed in cell type-specific fashion; LR8Ϫ predominates in developing germ cells and LR8ϩ in the accompanying somatic cells, i.e. granulosa and Sertoli cells, respectively (17, 18, 26) . In ligand blots, both forms were shown to react with mammalian RAP (18). Recently, we have obtained preliminary evidence for expression of RAP or a RAP-like protein in the chicken (27). To investigate the relevance of RAP expression in correlation to that of the variant receptor forms and in regard to the information on RAP in nonmammalian species, we have initiated studies to identify and molecularly and functionally characterize the first nonamniotic RAP.