Collagen-mediated platelet aggregation. Effects of collagen modification involving the protein and carbohydrate moieties.

In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the alpha1(I) and alpha2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 muM and 55 muM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 muM alpha1(I) and 160 muM alpha2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen. Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. One class of higher molecular weight contained sialic acid, glucosamine, galactosamine, fucose, mannose, galactose, and glucose, and the other of lower molecular weight consisted primarily of a mixture of galactose and galactosyl-glucose units O-glycosidically linked to hydroxylysine-containing peptides. We found that, after the residual tryptic activity contaminating the higher molecular weight fraction was inhibited, neither of the glycopeptide classes produced nor inhibited native human skin insoluble collagen-mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet-rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosylglucose (Hyl-Gal and Hyl-Gal-Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet-rich plasma showed that, at concentrations greater than 50 nM, Hyl-Gal gives apparent binding to platelets, but there was no evidence of Hyl-Gal-Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl-Gal (at 29 muM) nor Hyl-Gal-Glc (at 18 muM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 muM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Gal-Glc-containing 36 residue rat skin soluble collagen alpha1(I)cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Modification of at least 90% of the rat skin soluble collagen carbohydrate by mild periodate oxidation had no effect on the platelet aggregating activity. Human skin insoluble collagen was reacted with periodate under the same conditions, and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation.Thus, collagen-platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple-helical fold or perhaps even for a particular amino acid sequence in the triple helix.

A functional transmembrane complex: the luteinizing hormone receptor with bound ligand and G protein.

The luteinizing hormone receptor (LHR) is one of eight members in a cluster of the rhodopsin family of the large G protein-coupled receptor (GPCR) superfamily that contains some 800-900 genes in the human genome. LHR, along with its paralogons, follicle stimulating hormone receptor (FSHR) and thyroid stimulating hormone receptor, form one of the three classes in this cluster; the two other classes contain the relaxin-binding GPCRs and orphan GPCRs. These GPCRs are characterized by a relatively large ectodomain (ECD) containing leucine-rich-repeats (LRRs); in the class of glycoprotein hormone receptors, the LRR region is capped by N-terminal and C-terminal cysteine-rich regions. Binding of human chorionic gonadotropin (hCG) or luteinizing hormone to the LHR-ECD triggers a conformational change of the transmembrane region of the receptor facilitating binding and activation of Gs, followed by effector enzyme activation and subsequent intracellular signaling. Viewing LHR as a transmembrane anchoring protein that sequentially binds hCG and Gs to give the hCG-LHR-Gs complex, numerous interactions and conformational changes must be considered. There is, unfortunately, a paucity of structural data on LHR, but crystal structures exist for hCG, the homologous FSH-FSHR-ECD (N-terminal fragment) complex, rhodopsin (in the inactive state), an active form of Galphas (transducin), and the betagamma heterodimer. Using a combined experimental (site-directed mutagenesis followed by characterization in transfected cells) and computational (homology modeling and molecular dynamics simulations) approach, good working models are being developed for the protein-protein interaction faces and, in some cases, the ensuing conformational changes induced by complex formation. hCG binding to the LHR-ECD appears to involve several LRRs; LHR activation can be described in terms of disrupting a network of H-bonds in the cytosolic halves of helices 1-3, 6, and 7; and binding of LHR to Gs involves, in large part, intracellular loop 2 binding, presumably to Gsalpha at its C-terminus. Major gaps exist in our understanding at the molecular level of the six-polypeptide chain complex, hCG-LHR-Gs, but considerable progress has been made in the past few years.

Cross-reactivity of a monoclonal antibody directed against an estrogen-induced protein in MCF-7 human breast cancer cells with murine Leydig cell tumor proteins.

An estrogen-responsive murine Leydig cell tumor (M5480A) was examined for the presence of cross-reactive proteins to a monoclonal antibody directed against a Mr 24,000 estrogen-regulated protein in human breast cancer cells. Human breast tumor biopsies were used as controls for the cytosol preparations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot conditions used in these experiments. The estrogen-regulated Mr 24,000 protein was detected in sodium dodecyl sulfate-polyacrylamide gels of cytosols from four human breast tumor biopsies examined. Larger amounts of the Mr 24,000 protein were present in the two estrogen-progesterone receptor-positive tumor biopsies in comparison to the two estrogen-progesterone receptor-negative samples. In addition, the two receptor-positive samples demonstrated an additional, less intense immunoreactive band at Mr 21,000. Under identical conditions, the same monoclonal antibody bound to two major protein bands from sodium dodecyl sulfate-polyacrylamide gels of Leydig cell tumor cytosols at Mr 56,000 and Mr 86,000. Antibodies prepared from BALB/c mouse ascites fluid of animals bearing the parent myeloma cell line (P3X63NS1) exhibited no immunoreactivity against the human breast or Leydig cell tumor proteins. In light of the high degree of specificity which this monoclonal antibody exhibits, our results suggest that similar antigenic determinants may exist in these proteins from two distinct tumors.

Effect of 4-hydroxyandrostenedione on murine Leydig tumor cell steroidogenesis.

The murine Leydig cell tumor (M5480A) possesses high levels of estrogen receptor and is known to produce estrogens. In these studies we examined the effects of the potent aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) on Leydig tumor cell steroidogenesis both in vitro and in vivo. The addition of 4-OHA to Leydig tumor cells in primary culture resulted in a dose- and a time-dependent decrease in media progesterone levels. The observed decrease was most likely due to impaired synthesis of progesterone, inasmuch as no alteration in progesterone metabolism was seen when progesterone levels were diminishing. However, 4-OHA inhibited progesterone conversion to testosterone following 1 h of incubation, but this effect disappeared coincident with 4-OHA metabolism. Analysis of pregnenolone production revealed a biphasic dose-dependent effect of 4-OHA. At low doses (0.01-0.1 microM), 4-OHA was found to decrease pregnenolone concentrations, while at higher doses (1-10 microM) pregnenolone levels were elevated. Therefore, the actions of 4-OHA on Leydig cell steroidogenesis in vitro appear to be multifocal. Other experiments were performed to evaluate the effects of 4-OHA on tumor-bearing male mice in vivo. In these studies, the predominant effects of 4-OHA were to act as an aromatase inhibitor and to inhibit progesterone production. Thus, while 4-OHA is a potent aromatase inhibitor, we have found that this compound may alter steroidogenesis in Leydig tumor cells at several sites prior to aromatization.

Inhibition of Leydig tumor cell steroidogenesis by 10-propargylestr-4-ene-3,17-dione, an irreversible aromatase inhibitor.

The murine Leydig cell tumor (M5480A) was assayed for the presence of aromatase activity and for the effects of 10-propargylestr-4-ene-3,17-dione (PED), an aromatase inhibitor, on steroidogenesis. Microsomal preparations from these tumors contained low levels of aromatase activity which was PED sensitive. In addition, these Leydig tumor cells were maintained in primary culture and incubated under basal and gonadotropin-stimulated conditions with various doses of PED. Medium levels of progesterone, a major product of these cells, were found to decrease in a dose- and time-dependent manner upon addition of PED. To determine whether the observed effect was due to reduced synthesis or to increased metabolism of progesterone, tritiated progesterone was added to these cell cultures. Analysis of culture medium by high-performance liquid chromatography suggested that PED dramatically reduced the conversion of labeled progesterone to testosterone. Furthermore, examination of medium pregnenolone levels revealed diminished amounts of this steroid as well. Taken together, these results suggest that PED or its metabolites inhibit Leydig tumor cell steroidogenesis at several sites. Thus, in addition to its role as an aromatase inhibitor, this agent also has effects prior to pregnenolone production, as well as other effects in the pathway between progesterone and testosterone.

Preparation and characterization of a novel group of pituitary-derived peptides stimulatory for DNA synthesis in fibroblasts.

Using [3H]thymidine incorporation into Balb/c 3T3 fibroblasts as an index of mitotic activity, several cationic growth-promoting peptide fractions of relatively low potency were identified and partially purified from bovine pituitaries. The most completely characterized fraction exhibited an isoelectric pH of 10.4, and gel exclusion chromatograms under non-denaturing conditions were consistent with a self-associating system. Gel exclusion chromatography in the presence of 6 M guanidine hydrochloride demonstrated that the major reduced and S-alkylated peptides comprising this fraction had molecular weights of 8400 and 9000, and there was no evidence of disulfides between these or other minor peptides. This was confirmed by polyacrylamide gel electrophoresis in the presence of sodium lauryl sulfate and urea. Two other active fractions, each with an isoelectric pH of 9.9, were found to have major reduced and S-alkylated peptides with molecular weights between 6400 and 13 000, and the chromatographic results suggested the presence of intramolecular disulfides. The peptides from all fractions had a relatively high percentage of basic amino acids (approx. 20%); lysine was predominant (10-17%) and histidine was relatively low (less than 1%). Based on various criteria including amino acid composition, molecular weight, isoelectric point, and biological assays, the mitogenic activity in these fractions does not appear to result from fibroblast growth factor. Thus, the pituitary seems to contain multiple cationic mitogenic peptides of low potency for fibroblasts in vitro and their target cells in vivo remain to be defined.

Testicular membranes with improved stability of the gonadotropin receptor.

A plasma membrane fraction has been prepared from rat testis using an aqueous double-phase polymer system containing dextran, poly(ethylene glycol) 6000 and Zn2+. The membrane-associated gonadotropin receptor for lutropin and human choriogonadotropin can be markedly stabilized by a thawing-washing step of frozen membranes which prolongs the apparent half-life of the unoccupied membrane-associated receptors from less than 1 h at 37 degrees C to greater than 5h. Also, no degradation of 125I-labeled human choriogonadotropin was detected following incubation with the membrane fraction. The equilibrium binding was characterized by an apparent association constant of 1.6 x 10(10) M-1 and a receptor content of 33 fmol/mg protein. Binding kinetics yielded as association rate constant of 1.0 x 10(8) M-1 x min-1, while the dissociation rate constant for human choriogonadotropin was too low to be accurately determined under the conditions used. In contrast, ovine lutropin could be reversibly bound to the membranes leaving the previously occupied receptors available for binding by 125I-labeled human choriogonadotropin.

Characterization of protein tyrosine kinase activity in murine Leydig tumor cells.

The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.

Demonstration of distinct forms of testicular adenylate cyclases associated with germinal and Leydig cell fractions.

Decapsulated testes from adult rats were digested with collagenase, and the fraction enriched in germinal and Leydig cells was applied to a 0-4% continuous metrizamide gradient and centrifuged. This leads to separation of a germinal cell fraction and two putative Leydig cell populations that bind human choriogonadotropin, but only one of which responds to the gonadotropin with marked increase in testosterone production. Adenylate cyclase activity was present in these three fractions, and Mn2+ was more effective than Mg2+ as a divalent cation. The adenylate cyclase activity associated with the germinal cell fraction was just marginally stimulated by fluoride and by the non-hydrolyzable GTP analog 5'-guanylimidodiphosphate, while that associated with the Leydig cell populations was stimulated to a greater degree depending upon the type of divalent cation. Only the Leydig cell populations exhibited marked human choriogonadotropin-sensitive stimulation of adenylate cyclase activity in the presence of 5'-guanylimidodiphosphate above that observed with the GTP analog alone. These results suggest the presence of distinct adenylate cyclases in adult rat testis and indicate that both populations of Leydig cells are capable of producing cyclic AMP in response to gonadotropins such as human choriogonadotropin.

Characterization of the desensitized state of Leydig tumor cells.

A perifusion system has been used to study the in vitro desensitization of isolated Leydig tumor cells. It was observed that the cells become refractory, as measured by decreased rates of steroidogenesis, during continuous perifusions with saturating concentrations of either human choriogonadotropin (CG), cholera toxin, or 8-bromo-cyclic AMP. Furthermore, an initial perifusion of the cells with either human CG, cholera toxin, or 8-bromo-cyclic AMP causes subsequent desensitization towards all three stimuli. Thus, each of these stimuli is equally effective in inducing a state of desensitization in these cells that is manifested by a steroidogenic lesion(s) distal to cyclic AMP formation. It was found that the post-cyclic AMP lesion(s) in the desensitized state occurs prior to the formation of pregnenolone. However, the decreased rates of steroidogenesis do not seem to arise from a depletion of intracellular cholesterol.

Circular dichroism of glycopeptide fractions from alpha1-acid glycoprotein, thyroglobulin, and ovalbumin.

Using sequential pronase digestions, glycopeptide fractions were prepared from human alpha1-acid glycoprotein, hen egg ovalbumin, and bovine thyroglobulin, two types of glycopeptides being obtained from the latter. The fractions were characterized on the basis of hexose, hexosamine, sialic acid, and peptide content. The glycopeptide fraction from alpha1-acid glycoprotein is complex (i.e., the carbohydrate moiety contains mannose, galactose, N-acetylglucosamine, and sailic acid), as is one of the glycopeptide fractions from thyroglobulin (type I). The glycopeptide fraction from ovalbumin and the type II glycopeptide fractions from thyroglobulin are simple (i.e., the carbohydrate moiety contains only mannose and N-acetylglucosamine). The circular dichroic spectra of the two complex glycopeptide fractions and the ovalbumin glycopeptide fraction were similar and were characterized by a negative extremum between 207.5 nm and 211 nm with magnitudes in the range of --6400 deg-cm2-dmol-1 to --7200 deg-cm2-dmol-1 (referred to the molar concentration of N-acetylated sugars). The thyroglubulin type II glycopeptide fraction exhibited a circular dichroic spectrum with an extremum of --29 200 deg-cm2-dmol-1 at 205 nm. Removal of sialic acid from the complex glycopeptide fractions greatly increased the (negative) magnitude of ellipticity at the extremum. The circular dichroic spectra of the complex of glycopeptide fractions were reasonably additive using the spectra of monomeric sialic acid and the asialo-derivatives. This demonstrates that the contributions of sialic acid to the circular dichroic spectrum are nearly additive. The implications of this observation are that covalent attachment of these terminal residues to the oligosaccharides does not lead to strong interactions with other chromophores nor to positioning in particularly asymmetric environment. In contrast, the magnitudes of the observed ellipticity extrema in the circular dichroic spectra of the asialo-derivatives, in which N-acetylglucosamine is the major chromophore, are much greater than can be accounted for on the basis of monomeric contributions (i.e., free N-acetylglucosamine). This finding shows that the optical activity of N-acetyl-glucosamine is greatly influenced by the formation of the carbohydrate core in glycoproteins and suggests the possible formation of secondary structure in the carbohydrate moiety.

Evidence for structural dissociation of two biologic actions of growth hormone.

The effects of purified growth hormone and its CNBr fragments on somatomedin induction and on the stimulation of hepatic and renal ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) activity in rats have been investigated. At the doses tested, none of the CNBr fragments induced somatomedin as evidenced by lack of an effect on sulfate, leucine, and thymidine incorporation into cartilage of hypophysectomized rats. However, the largest fragment, consisting of two peptides corresponding to Residues 6-124 and 150-179 linked by a disulfide bridge, stimulated both renal and hepatic ornithine decarboxylase activity in hypophysectomized rats and the activity of the hepatic enzyme in intact animals. A smaller CNBr fragment corresponding to Residues 125-149 slightly stimulated the activity of renal ornithine decarboxylase but failed to increase activity of the hepatic enzyme. A similar slight stimulation of the activity of the renal, but not the hepatic, enzyme was produced by a large carboxyl-terminal fragment (molecular weight 8000) prepared by proteolytic cleavage of partially purified ovine growth hormone. Circular dichroic spectra of the CNBr fragments demonstrated that the largest fragment retained much of the ordered secondary structure of intact growth hormone while two smaller CNBr fragments were devoid of ordered secondary structure. These observations indicate that different biological activities of growth hormone may be dissociated by fragmentation of the parent molecule.

Endothelial and myocardial injury during ischemia and reperfusion: pathogenesis and therapeutic implications.

Early reperfusion remains the most effective way of limiting myocardial necrosis and improving ventricular function in experimental models and human patients. However, the introduction of oxygen and cellular elements, especially the neutrophil, into the ischemic zone may initiate a deleterious cascade of events that limits myocardial salvage after reperfusion. Although the pathogenesis of reperfusion injury remains controversial, recent studies have suggested that the endothelium may play a critical role. Endothelial cells maintain flow in the microcirculation by secreting a number of vasodilatory compounds and substances that prevent plugging of capillaries by inhibiting neutrophil adherence and platelet aggregation. Reperfusion of ischemic myocardium accelerates structural and functional changes in endothelial cells, resulting in a progressive decrease in microcirculatory flow ("no reflow" phenomenon). Numerous studies suggest that activated neutrophils mediate vascular damage by releasing reactive oxygen species and potent proteolytic enzymes. The administration of therapeutic agents that limit endothelial disruption and neutrophil plugging has shown promising results in limiting myocardial reperfusion injury in experimental models.

Beneficial long-term effect of intracoronary perfluorochemical on infarct size and ventricular function in a canine reperfusion model.

The administration of a drug soon after reperfusion that could enhance myocardial salvage would have important clinical application. The aim of this study was to assess the long-term effect of the perfluorochemical, Fluosol DA 20%, on infarct size, infarct morphology, ventricular ectopic activity and serial regional ventricular function in a 2 week closed chest canine model. After 90 minutes of proximal left anterior descending artery occlusion, animals randomly received either oxygenated Fluosol DA (n = 9) or saline solution (n = 9) intracoronary at 15 ml/kg body weight over 20 to 30 minutes. Hemodynamic variables were similar in the two groups except for transient elevation of left ventricular filling pressure immediately after infusion in the treated group. Infarct size was markedly reduced in the perfluorochemical-treated animals when expressed as a percent of the risk region (10.8 +/- 1.8% versus 28.9 +/- 5.5%, p less than 0.02) or as a percent of the total left ventricle (3.7 +/- 1% versus 10.8 +/- 8%, p less than 0.006). This was associated with greater improvement in radial shortening in the jeopardized zone at 2 weeks after reperfusion (15.3 +/- 2.8% versus 5.2 +/- 2.1%, p less than 0.01). Histologic examination revealed adequate healing in the treated animals with an increased number of swollen mononuclear cells in the border zones. Holter electrocardiographic recordings demonstrated a low frequency of ventricular ectopic beats in both groups. This study suggests that the perfluorochemical, Fluosol DA, may be a potentially useful agent in enhancing myocardial salvage after successful reperfusion.

The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin.

The glycoprotein hormones are heterodimeric and contain a common alpha-subunit, which is noncovalently associated with a hormone-specific beta-subunit. The alpha-subunit has been highly conserved throughout evolution; for example, the five amino acid residues of the carboxy-terminus, Tyr-Tyr-His-Lys-Ser-COOH, are identical in nine of the 10 available amino acid sequences. It has been shown that enzymatic removal of these five amino acid residues, while not affecting holoprotein formation, results in a heterodimer that exhibits very little, if any, binding to the CG/LH receptor. Using site-directed mutagenesis on the human alpha-subunit, we have prepared two deletion mutants, Des-(88-92)alpha and Des-(89-92)alpha, and two point mutants, where each of the two tyrosines, 88 and 89, was replaced with phenylalanine, in order to delineate more specifically the contributions of these aromatic side-chains to receptor binding. The cDNAs for wild-type hCG alpha and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCG beta was inserted into a pRSV plasmid; both were transiently cotransfected into DUXB-11 cells. The media were collected, and RIAs showed that all mutants formed heterodimers; moreover, there was no discernable difference in subunit assembly between wild-type hCG alpha and the various mutant alpha-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [125I]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional expression of yoked human chorionic gonadotropin in baculovirus-infected insect cells.

hCG is a glycoprotein hormone composed of an alpha-subunit, common to all gonadotropins and to TSH, and a hormone-specific beta-subunit. The non-covalent association of the two subunits is an obligatory step for the formation of biologically active hormones. The correct assembly of the heterodimer is also important for efficient secretion of the hormone, receptor binding, and signal transduction. Herein, we have demonstrated that expression of the two subunits from independent promoters present in a single recombinant baculovirus resulted in subunit association and secretion of biologically active holoprotein by the insect cells. To determine whether the active conformation of heterodimer could be achieved when the two subunits were synthesized in tandem on a single polypeptide chain, two single chain or yoked hCG1, the C-terminus of the complete beta-subunit (145 amino acid residues) was conjoined to the N-terminus of the alpha-subunit. Yoked hCG2 was similar, except that it contained the N-terminal 123 amino acid residues of the beta-subunit. Both yoked hCG molecules bound LH/CG receptor with high affinity and stimulated adenylate cyclase and progesterone levels in transformed mouse Leydig (MA-10) cells. Therefore, the alpha- and beta-subunits are able to fold into a biologically active conformation when covalently linked. Interestingly, when compared with urinary hCG, the hormone expressed in baculovirus-infected insect cells binds to the LH/CG receptor with higher affinity, but exhibits diminished signaling, thus providing another example of a partial dissociation between receptor binding and activation.

Holoprotein formation of human chorionic gonadotropin: differential trace labeling with acetic anhydride.

The effects of holoprotein formation in human CG (hCG) on the reactivities of several of the individual amino groups of each subunit were investigated by differential trace labeling with [3H]acetic anhydride. The alpha- and beta-subunits were labeled separately, as was hCG, under conditions chosen to ensure that an average of less than one amino group was modified per molecule. Although the beta-subunit contains fewer amino groups than the alpha-subunit, most of the 3H incorporation occurred in beta at the N-terminal region. Chemical and enzymatic cleavage of the subunits enabled us to identify several individual amino groups and, from measurements of the incorporated radioactivity of the free subunits and intact hormone, determine their protection factor, which is a measure of the reactivity and thus of the local environment and changes thereof upon holoprotein formation. Lys51 and Lys91 of alpha were approximately 2-fold more reactive and less reactive, respectively, in the alpha beta complex than in the free subunit. The alpha-amino group of alpha was characterized by comparable reactivities in the heterodimer and free subunit, as was Lys44/Lys45 when analyzed as a pair; the reactivity of alpha-Lys44 was slightly less in the holoprotein than in the free subunit. The alpha-amino group and Lys2 of beta could not be resolved by available cleavage procedures; consequently they were analyzed as a pair and found to be some 2-fold less reactive in the heterodimer than in the free subunit, as was Lys104 of beta. From these results, we can conclude that subunit assembly produces changes in the microenvironments of several amino groups, attributable to steric effects, specific intermolecular interactions, and localized conformational changes. Analysis of these data with reference to the recently determined crystal structure of hydrogen fluoride-treated hCG enabled a distinction to be made of these possibilities for several of the amino groups.

Transiently elevated levels of c-fos and c-myc oncogene messenger ribonucleic acids in cultured murine Leydig tumor cells after addition of human chorionic gonadotropin.

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta.

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.

Functional role of transmembrane helix 7 in the activation of the heptahelical lutropin receptor.

A member of the G protein-coupled receptor superfamily, the LH receptor (LHR), and the two other glycoprotein hormone receptors are distinguished from the other members by the presence of a relatively large N-terminal extracellular domain that is responsible for high-affinity ligand binding. Transmembrane helix (TMH) 7 of LHR is amphipathic, with an extended face containing only hydrophobic side chains and another containing both hydrophobic and polar side chains with potential hydrogen bond donor and acceptor functions. Since several reports have shown the importance of this helix in ligand-mediated signaling, we have used Ala scanning mutagenesis to study eight amino acid residues of rat LHR that are invariant in the three glycoprotein hormone receptors, Leu586, Val587, Asn593, Ser594, Cys595, Asn597, Phe604, and Thr605. The wild type (WT) and mutant cDNAs were transiently transfected into COS-7 cells for characterization by human CG (hCG) binding and cAMP production. No differences were detected in dissociation constants (K(d)S) or basal cAMP production relative to WT LHR, but three categories of LHR mutants were distinguished from WT LHR based upon their expression levels and responsiveness to hCG: 1) comparable or higher expression but reduced ligand responsiveness (N593A and C595A), 2) reduced expression and ligand responsiveness (N597A and T605A), and 3) comparable expression and responsiveness (L586A, V587A, S594A, and F604A). Three other mutants, C595M, F604Y, and T605Y, were comparable to WT LHR in ligand responsiveness. To provide more information on Asn593 and Asn597, a total of 12 replacements were investigated. Of considerable interest and potential significance was the finding that many of the replacements in LHR resulted in either loss of function (N593A, Q, S; N597R) or gain of function (N593R and N597Q), this being the first evidence of a position in LHR that, depending upon the nature of the amino acid residue, can result in constitutive activation and/or diminished responsiveness to ligand. The results of molecular modeling and energy minimization of TMHs 6 and 7, based on a postulated interaction between Asp556 (TMH 6) and Asn593/Asn597 (TMH 7), indicated that, while there is not a correlation between function and predicted energies of WT LHR and the mutants, reorientation of one or both helices is responsible for the functional changes observed. Possible interactions of TMHs 3 and 4 and of 5 and 6 were suggested by molecular modeling. Ten mutants were prepared of two amino acid residues that are invariant in the glycoprotein hormone receptors and have side chain hydrogen bond donor and acceptor function, Glu429 in TMH 3 and Asn513 in TMH 5. Expression levels and hCG-mediated signaling were reduced in most of the LHR mutants, but none of these exhibited constitutive receptor activation. We conclude that Glu429 is not critical for receptor function, while Asn513 appears to be particularly important in receptor folding and/or trafficking. The results reported herein indicate an important role for TMH 7, and particularly Asn593 and Asn597, in the process of receptor activation. Moreover, these two asparagines, although in close proximity to each other in TMH 7, are quite distinct in function as evidenced by certain replacements that can lead to loss of function in one and gain of function in the other.