The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the Kd of ICAM-1 binding. The Kd of wild-type LFA-1 (1.2 × 10−1 s−1) differed with that of the activating IDAS mutant (1.9 × 10−2 s−1), but their Ka values were identical (2.2 × 105 M−1s−1). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.

The findings from this study confirm the role of diet-related exposure in the etiology of gastric cancer from the point of view of epidemiology. An increased risk of gastric cancer is associated with high intakes of protein, saturated fat, cholesterol and sodium, while consumption of polyunsaturated fat, vitamin A and ascorbic acid may have a protective effect against gastric cancer.

The amino-terminal region of the humnn sex steroid-binding protein of plasma (SBP or SHBG) containing Kl34 and MI 39 was found to rcprcsent part of the steroid-binding site. This was aceomplishcd by constructing and expressing site-directed mutants having the following replacements: MI39L, Ml39K. M139S, Kl34A, H235S, and Y57F. The results indicated that Ml39L and H23SS were fuily-active, Kl34A and YS7F wcrc 50 and G7% active, Ml39K was 7% active, and M 139s was inactive. These results support affinity-labeling data indicating that both K134 and Ml39 are located in or near the site, and suggest that Y57 may play a role in skroid binding. The fully active H23SS mutant reveals thnt H235 is not involved in the steroid-binding process.

Complete dissociation of dimeric plasma sex steroid-binding protein (SBP or SHBG) was obtained in 6 M urea at 10 "C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca++ or steroid. Renatured monomers yield dimers with dissociation constants for Sa-dihydrotesterone (DHT) and 17a-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric steroid-binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds steroid can be considered meaningless; steroid-binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.

A M-length 1,209 bp cDNA encoding the human sex steroid-binding protein of plasma (SBP or SHBG) and testis (ABP) was cons~ructi and expressed in BHK-21 cells. The sequence agrees with the published gene and protein sequences. The cells were found to secrete SBP following transfection and G418' selcctior:. The recombinant protein binds Sol-dihydrotestosteronc with a Kd of 0.28 nM. It also binds testosterone and 17b-cstradiol but not progesterone, estrone or cortisol revealing a steroid-binding sjxciiicity identical to that of human SBP. SDS-PAGE patterns are less complex than human SBP and show a monomeric molecular weight of about 43 kDa.Sex steroid-binding protein; SBP, SHBG; Mammalian expression