Structure-function relationships of rat androgen--binding protein/human sex-hormone binding globulin: the effect of mutagenesis on steroid-binding parameters.
Androgen-binding protein (ABP) and plasma sex hormone-binding globulin (SHBG) are encoded by the same gene and have the identical amino acid sequence within a species. Mammalian ABPs and SHBGs bind sex steroids with high affinity, but some binding properties differ among species. Human SHBG has a higher affinity for steroids and forms a more stable 5 alpha-dihydrotestosterone (DHT) complex (t1/2 = 40 min) than rat epididymal ABP (t1/2 = 5 min) at 0 C. In this study it was found that recombinant wild-type rat ABP/SHBG bound DHT and estradiol with nearly the same affinities as human SHBG, rather than the affinities of rat epididymal ABP. This finding is reminiscent of our previously published data demonstrating that ABP secreted by cultured Sertoli cells had a higher affinity for DHT than did epididymal ABP. Recombinant wild-type ABP had DHT dissociation properties similar to those of rat epididymal ABP. The differences in binding properties of epididymal ABP and recombinant ABP could in part be caused by intrinsic differences in the properties of the proteins due to posttranslational modifications or allelic variations in sequence. To aid in identification of the steroid-binding domain of ABP and SHBG, we developed recombinant rat ABP/SHBG chimeras containing human sequences in and flanking the putative steroid-binding region (residues 134-150). Four regions were mutated: 1) residues 129-132; 2) residues 133-138; 3) residues 148-155; and 4) 165-169; residues between these regions are identical in rat and human ABP/SHBG. Wild-type (ABPwt) and mutant proteins were expressed in COS cells, and their steroid-binding properties were determined. Conversion of rat amino acid residues 133-169 (ABP2,3,4) to human ABP/SHBG sequence resulted in a 2-fold increase in affinity for estradiol compared to ABPwt. Another mutant ABP2,3,4; Leu 137, which contained the rat Leu137 residue, had a 5-fold increase in estradiol affinity. Conversion of residues 129-132 to human sequence in ABP2,3,4 to form ABP1,2,3,4 resulted in a dramatic decrease in estradiol affinity. Conversion of each region separately also resulted in some changes in steroid-binding properties. The largest change was observed with ABP1, which had a 3-fold reduction in estradiol affinity compared to ABPwt. There was a 14-fold difference in estradiol affinity between ABP1 and ABP2,3,4; Leu 137. Alterations of some individual amino acid residues in region 2, which is the least conserved region between rat and human, caused subtle or major changes in the estradiol-binding properties; none affected DHT binding.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633.
B J
BJ
Danzo
D R
DR
Joseph
D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D002678:Chimera; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015345:Oligonucleotide Probes / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013256:Steroids / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000727:Androgen-Binding Protein; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin; D013256:Steroids