Structure/function analyses of human sex hormone‐binding globulin by site‐directed mutagenesis

Abstract

Human sex hormone-binding globulin (hSHBG) and rat androgen-binding protein (rABP) exhibit distinct afilnities for sex-steroids. We therefore constructed and expressed a hSHBG/rABP hybrid cDNA encoding the N-terminal portion of hSHBG (205 residues) and the C-terminal portion of rABP (168 residues). The resulting chimera displayed similar steroid-binding characteristics as hSHBG and was recogniscd by a monoclonal antibody (SlB5) for hSHBG. We then created substitutions at Ser-133, His-136 and Met-139. The Asp-133 and Gin-136 mu*zmts bound steroids in the same way as normal hSHBG while the steroid-binding aflinity of Trp-I 39 was reduced. All three mutants cross-reacted similarly in a hSHBG radioimmunoassay, but Gin-136 was recognised poorly by the Sl BS antibody. Thesedata imply that residues involved in steroid-binding are located within the N-terminal half of hSHBC and include Met-139, and that the SIB5 epitopc is located in this region.

Photoaffinity Labeling of Homologous Met-133 and Met-139 Amino Acids of Rabbit and Sheep Sex Hormone-Binding Globulins with the Unsubstituted Δ6-Testosterone Photoreagent

“…Site-directed mutagenesis experiments (24)(25)(26)(27) have confirmed the importance of the Met-139 residue for steroid binding by human SHBG and have demonstrated that the steroid-binding site is mainly situated within the Leu-1-Glu-205 portion of human SHBG which comprises the above-described sites of affinity labeling.…”

Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli

“…A human SHBG cDNA in the pSELECT-1 vector (Promega) was mutated using an oligonucleotide primer (according to the protocol provided by Promega) to introduce a BamHI site immediately 5' to the codon for Leu-1 in the mature polypeptide (Hammond et al, 1987;Bocchinfuso et al, 1992b). The resulting cDNA was then excised with BamHI and inserted into a BamHI-digested pGEX-2T vector (Pharmacia) to create a cDNA that encodes a GST/SHBG fusion protein, designated SHBG( 1-373) as shown in Table 1.…”

Sex hormone-binding globulin is synthesized in target cells

“…This question arises because, as we shall review, it is clear that SHBG is synthesized in these two tissues. Two major SHBG transcripts are known, each originating from a different promoter (minor SHBG transcripts have received little attention and will not be discussed here) (Gershagen et al 1987, 1989, 1991, Hammond et al 1987, 1989, Joseph et al 1991, Bocchinfuso et al 1992, Bocchinfuso & Hammond 1994, Hammond & Bocchinfuso 1996, Janne et al 1998. The first major transcript encodes a precursor for the secreted (plasma) form of SHBG, and was originally described in the liver (SHBG L ) (Que & Petra 1987), while the second encodes a protein of unknown function and was originally described in the testis (SHBG T ) (Hammond et al 1989).…”