Lysophosphatidic acid (LPA) is a serum-borne phospholipid that exerts a pleiotropic range of effects on cells through activation of three closely related G-protein-coupled receptors termed LPA1/EDG-2,LPA2/EDG-4 and LPA3/EDG-7. Of these receptors, the LPA1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA1receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. Treatment with LPA induced the rapid endocytosis of approximately 40%of surface LPA1 within 15 minutes. Internalization was both dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removal of agonist following 30 minutes incubation resulted in recycling of LPA1 back to the cell surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). In addition, both dynamin2 K44A and Rab5 S34N mildly inhibited LPA1-dependent activation of serum response factor. Finally, our results also indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.

The human type 1 (placenta, breast tumors, and prostate tumors) and type 2 (adrenals and gonads) isoforms of 3␤-hydroxysteroid dehydrogenase/isomerase (3␤-HSD1 and 3␤-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern. Our recent studies have shown that His 156 contributes to the 14-fold higher affinity that 3␤-HSD1 exhibits for substrate and inhibitor steroids compared with human 3␤-HSD2 containing Tyr 156 in the otherwise identical catalytic domain. Our structural model of human 3␤-HSD localizes His 156 or Tyr 156 in the subunit interface of the enzyme homodimer. The model predicts that Gln 105 on one enzyme subunit has a higher probability of interacting with His 156 on the other subunit in 3␤-HSD1 than with Tyr 156 in 3␤-HSD2. The Q105M mutant of 3␤-HSD1 (Q105M1) shifts the Michaelis-Menten constant (K m ) for 3␤-HSD substrate and inhibition constants (K i ) for epostane and trilostane to the much lower affinity profiles measured for wild-type 3␤-HSD2 and H156Y1. However, the Q105M2 mutant retains substrate and inhibitor kinetic profiles similar to those of 3␤-HSD2. Our model also predicts that Gln 240 in 3␤-HSD1 and Arg 240 in 3␤-HSD2 may be responsible for the 3-fold higher affinity of the type 1 isomerase activity for substrate steroid and cofactors. The Q240R1 mutation increases the isomerase substrate K m by 2.2-fold to a value similar to that of 3␤-HSD2 isomerase and abolishes the allosteric activation of isomerase by NADH. The R240Q2 mutation converts the isomerase substrate, cofactor, and inhibitor kinetic profiles to the 4 -14-fold higher affinity profiles of 3␤-HSD1. Thus, key structural reasons for the substantially higher affinities of 3␤-HSD1 for substrates, coenzymes, and inhibitors have been identified. These structure and function relationships can be used in future docking studies to design better inhibitors of the 3␤-HSD1 that may be useful in the treatment of hormone-sensitive cancers and preterm labor.The human type 1 (placenta, mammary gland, and prostate) and type 2 (adrenals, ovary, and testis) isoforms of 3␤-hydroxysteroid dehydrogenase (EC 1.1.1.145)/steroid ⌬ 5 -⌬ 4 -isomerase (EC 5.3.3.1) (3␤-HSD1 1 and 3␤-HSD2) are encoded by two distinct genes that are expressed in a tissue-specific pattern (1). As shown in Fig. 1, human 3␤-HSD1 catalyzes the conversion of 3␤-hydroxy-5-ene-steroids (dehydroepiandrosterone (DHEA) and pregnenolone) to 3-oxo-4-ene-steroids (androstenedione and progesterone), and human 3␤-HSD2 converts 17␣-hydroxypregnenolone and pregnenolone to ultimately produce cortisol and aldosterone in the human adrenal, respectively (2). 17␣-Hydroxylase/17,20 lyase (CYP17) in the human adrenal gland converts pregnenolone to DHEA, which is the major circulating steroid in humans as DHEA sulfate (2). In placenta, androstenedione is converted by aromatase and 17␤-hydroxysteroid dehydrogenase (17␤-HSD) to estradiol, which participates in the cascade of events that initiates labor in humans (2, 3). Placental 3␤-HSD1 also converts pregnenolone to...

Mammalian 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3β-HSD type 1 (3β-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17β-hydroxysteroid dehydrogenase (17β-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3β-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3β-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3β-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions respectively.

Human 3 -hydroxysteroid dehydrogenase/isomerase (3 -HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17 -hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3 -HSD reaction. Our three-dimensional homology model of 3 -HSD shows that Tyr154 and Lys158 are oriented near the 3 -hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3 -hydroxysteroid dehydrogenase/ isomerase (3 -HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3 -HSD activity and has a K m value (83·6 µM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3 -HSD activity (DHEA K m =11·2 µM; k cat =0·8 min -1 ) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K m value (27·6 µM) that is almost identical to wild-type. The K m value (4·3 µM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3 -HSD and NADH as the activator for isomerase with K m values that are similar to wild-type. The 3 -HSD activities of S123A and wild-type 3 -HSD increase by 2·7-fold when the pH is raised from 7·4 to the optimal pH 9·7, but S124A exhibits very low residual 3 -HSD activity that is pH-independent.These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3 -HSD activity. Since there are 29 Ser residues in the primary structure of human 3 -HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3 -HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3 -HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.