Mutagenesis of essential functional residues of rat androgen-binding protein/sex hormone-binding globulin.
Testicular androgen-binding protein (ABP) and liver (plasma) sex hormone-binding globulin (SHBG) are extracellular carrier proteins that bind androgens with high affinity. Both proteins are encoded by the same gene and have the same primary amino acid sequence. Previous affinity labeling experiments to identify the steroid-binding site of ABP/SHBG led to ambiguous results, implicating various residues from 134 to near the C-terminus. To aid in elucidation of the essential functional residues of ABP/SHBG, we created mutant rat proteins by deletion and site-directed mutagenesis. The mutants were expressed in COS 7 green monkey kidney cells and analyzed for immunoreactive cellular and medium ABP and dihydrotestosterone (DHT) binding properties. Analysis of truncated ABP proteins revealed that removal of 26 or more residues from the C-terminus eliminates secretion and DHT-binding activity. Alteration of amino acid residues by site-directed mutagenesis from residue 54 to residue 333 resulted in elimination of DHT binding for 9 of 10 mutants and reduced DHT affinity for one altered protein (ABPGly54-57). Only one of the 10 mutant ABP proteins was secreted by the COS cells. This secreted mutant ABP (ABPArg139) exhibited no detectable DHT-binding activity. Thus, our data demonstrate that modifications of the ABP primary sequence throughout the molecule have a detrimental effect on steroid binding and secretion. These data, taken together with previous affinity labeling experiments, mutagenesis studies, and the conserved residues between rat and human ABP/SHBG, indicate that at least part of active site is located in residues 139-150, but most of the protein is required to maintain the conformation of the active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Joseph
D R
DR
Department of Pediatrics, University of North Carolina, Chapel Hill 27599.
Lawrence
W
W
D000595:Amino Acid Sequence; D000727:Androgen-Binding Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D013196:Dihydrotestosterone / Q000378:metabolism; D017353:Gene Deletion; D008099:Liver / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D051381:Rats; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013737:Testis / Q000737:chemistry; D014162:Transfection
D000727:Androgen-Binding Protein; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; D004247:DNA