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The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.

The regulation of the sexually differentiated steroid sulfate 15 beta-hydroxylase, cytochrome P-450(15) beta of female rat liver has been investigated. Specific antibodies raised to isozyme P-450(15) beta were used with the Western blot technique to quantitate the specific levels of P-450(15) beta in liver microsomes. The method demonstrated that the levels of the protein are about 16-fold higher in female than in male microsomes and also showed that the specific microsomal content of P-450(15) beta is controlled by GH. Hypophysectomy of female animals resulted in a decrease of P-450(15) beta to male levels. Continuous infusion of human GH, mimicking the female pattern of GH secretion in intact male animals, caused an elevation of the P-450(15) beta level to that of the female. The same dose of human GH in hypophysectomized male or female animals raised the P-450(15) beta level 8-fold or 50% of that seen in normal females. Infusion of ovine PRL to intact male rats had no effect on P-450(15) beta levels, whereas infusion of rat GH caused a 4-fold increase. Thus, the regulation of P-450(15) beta by GH is mainly associated with the somatogenic properties of the hormone. Furthermore, sc injection of rat GH every 12 h, mimicking the male pattern of GH secretion, had no effect on P-450(15) beta levels, demonstrating the importance of the GH secretory pattern in regulation of the specific protein levels. Postpubertal castration of male animals did not influence the microsomal P-450(15) beta content, whereas neonatal castration led to a feminization of the P-450(15) beta content in the adult male rat. Administration of estradiol valerate to male animals caused complete feminization of P-450(15) beta levels, whereas administration of androgen to female animals caused a decrease to male levels. Before 21 days of age, the P-450(15) beta level was slightly higher in male than in female rats. At 35 days, however, the P-450(15) beta level in female rats had increased almost 100-fold, whereas the levels in males increased only slightly. These changes are concomitant with the development of the sexual differentiation of the GH secretory pattern, supporting the role of GH in P-450(15) beta regulation. In conclusion, isozyme P-450(15) beta is a GH-regulated enzyme specific for female rats. The low level of the protein in males is probably explained by neonatal androgenic programming of the GH secretory pattern.

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