NORMAL GENOMIC

• Tando Y, Kubokawa K
• Expression of the gene for ancestral glycoprotein hormone beta subunit inthe nerve cord of amphioxus.
• Gen Comp Endocrinol. 2009; 162: 329-39
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• Amphioxus belongs to the subphylum cephalochordata, a clade of chordatesphylogenetically placed at the most basal position. Despite many studieson the endocrine system of amphioxus, there were no confident lines ofevidence on the presence of pituitary hormones, whereas recent amphioxusgenome analysis reported that amphioxus has no pituitary hormone exceptfor thyrostimulin, which is a glycoprotein hormone in the pituitary,brain, and other organs of vertebrates. In the present study, we clonedcDNA for one glycoprotein hormone beta subunit (GPB) from amphioxus,AmpGPB5, and phylogenetically indicated that AmpGPB5 is the ancestralmolecule of glycoprotein hormone beta subunits of vertebrates includingpituitary glycoprotein hormones. Synteny analyses showed conservation ofchromosomal location of genes near GPB genes from amphioxus through human.The AmpGPB5 gene was expressed in a restricted region of the dorsal partof the nerve cord, glandular atrial cells of gills, and pre-vitellogenicoocytes in amphioxus. However, expression was not detected in theHatschek's pit which is considered to be a primitive pituitary gland. Onthe basis of present results, we hypothesize that a portion of vertebratepituitary hormones might be derived from an ancestral glycoprotein hormoneof amphioxus that functions as a neuroendocrine hormone.

• Thackray VG, Hunnicutt JL, Memon AK, Ghochani Y, Mellon PL
• Progesterone Inhibits basal and gonadotropin-releasing hormone inductionof luteinizing hormone beta-subunit gene expression.
• Endocrinology. 2009; 150: 2395-403
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• LH and FSH play critical roles in mammalian reproduction by mediatingsteroidogenesis and gametogenesis in the gonad. Gonadal steroid hormonefeedback to the hypothalamus and pituitary influences production of thegonadotropins. We previously demonstrated that progesterone differentiallyregulates the expression of the LH and FSH beta-subunits at the level ofthe gonadotrope: FSHbeta transcription is induced, whereas LHbeta isrepressed. In this study, we investigated the mechanism of progesteronerepression of LHbeta gene expression using immortalizedgonadotrope-derived LbetaT2 cells. The progesterone suppression of bothbasal and GnRH-induced LHbeta gene expression occurs in a hormone- andreceptor-dependent manner. Chromatin immunoprecipitation demonstrates thatthe hormone-bound progesterone receptor (PR) is recruited to theendogenous mouse LHbeta promoter. In addition, suppression requires boththe amino-terminal and DNA-binding regions of PR. Furthermore,progesterone suppression does not require direct PR binding to thepromoter, and, thus, PR is likely recruited to the promoter via indirectbinding through other transcription factors. These data demonstrate thatthe molecular mechanism for progesterone action on the LHbeta promoter isdistinct from FSHbeta, which involves direct PR binding to the promoter toproduce activation. It also differs from androgen repression of LHbetagene expression in that, rather than Sp1 or steroidogenic factor-1elements, it requires elements within -300/-250 and -200/-150 that alsocontribute to basal expression of the LHbeta promoter. Altogether, ourdata indicate that progesterone feedback at the level of the pituitarygonadotrope is likely to play a key role in differential production of thegonadotropin genes.

• Thackray VG, Mellon PL
• Synergistic induction of follicle-stimulating hormone beta-subunit geneexpression by gonadal steroid hormone receptors and Smad proteins.
• Endocrinology. 2008; 149: 1091-102
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• LH and FSH play crucial roles in mammalian reproduction by mediatingsteroidogenesis and gametogenesis. Gonadal steroid hormones influencegonadotropin production via feedback to the hypothalamus and pituitary. Wepreviously demonstrated that progesterone and testosterone can stimulateexpression of the FSH beta-subunit gene in immortalizedgonadotrope-derived LbetaT2 cells. Herein, we investigate how thesegonadal steroids modulate activin signaling in the gonadotrope.Cotreatment of LbetaT2 cells or mouse primary pituitary cells withsteroids and activin results in a synergistic induction of FSHbeta geneexpression. This synergy decreases when DNA-binding mutations areintroduced into the steroid receptors or when mutations that reducesteroid hormone responsiveness are introduced into the FSHbeta promoter,indicating that synergy requires direct DNA binding of the steroidreceptors. Furthermore, classical activin signaling via Smad proteins isnecessary for this synergy. In addition, these steroid receptorsphysically interact with Smads and are sufficient for the synergism tooccur on the FSHbeta promoter. Disruption of Smad binding to the promoterwith a Smad protein lacking the DNA-binding domain or an FSHbeta promotercontaining mutated activin-response elements prevents the synergisticenhancement of FSHbeta transcription. Collectively, our data demonstratethat the molecular mechanism for gonadal steroid hormone action on theFSHbeta promoter involves cross-talk between the steroid and activinsignaling pathways. They also reveal that this synergism requires bindingof both the steroid receptors and Smad proteins to their cognateDNA-binding elements and likely involves a direct protein-proteininteraction between the two types of transcription factors.

• Narayan P, Gray J, Puett D
• Yoked complexes of human choriogonadotropin and the lutropin receptor:evidence that monomeric individual subunits are inactive.
• Mol Endocrinol. 2002; 16: 2733-45
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• Human choriogonadotropin (hCG) contains an alpha-subunit, common to othermembers of the glycoprotein hormone family, and a unique beta-subunit thatdetermines hormone specificity. It is generally thought that heterodimerformation is obligatory for full hormonal activity, although other studieshave indicated that individual subunits and homodimeric hCGbeta werecapable of low affinity binding to the LH receptor (LHR) and subsequentactivation. Previously, we constructed two yoked hormone (hCG)-LHRcomplexes, where the two hormone subunits and the heptahelical receptorwere engineered to form single polypeptide chains, i.e. N-beta-alpha-LHR-Cand N-alpha-beta-LHR-C. Expression of both complexes led to constitutivestimulation of cAMP production. In the present study, we investigatedwhether the human alpha-subunit and hCGbeta can act as functional agonistswhen covalently attached to or coexpressed with the LH receptor. Ourinitial results showed that hCGbeta, but not alpha, was able to activateLHR with an increase in intracellular cAMP in human embryonic kidney 293cells but not in Chinese hamster ovary or COS-7 cells. Further examinationof this apparent cell-specific agonist activity of hCGbeta revealed thatlow levels of endogenous alpha-subunit were expressed in human embryonickidney 293 cells, thus enabling sufficient amounts of active heterodimerto form with the transfected hCGbeta to activate LHR. The studies inChinese hamster ovary and COS-7 cells clearly demonstrate that, even underexperimental conditions where hormone-receptor interactions are maximized,individual subunits of hCG can not act as functional agonists, at least intheir monomeric form.

• Fortenberry Y, Hwang JR, Apletalina EV, Lindberg I
• Functional characterization of ProSAAS: similarities and differences with7B2.
• J Biol Chem. 2002; 277: 5175-86
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• Prohormone convertases (PC) 1 and 2, enzymes found primarily inneuroendocrine tissues, are thought to mediate the proteolytic cleavage ofmany peptide precursors. To date, endogenous binding proteins for both PC2(7B2) and PC1 (proSAAS) have been identified. Although 7B2 represents apotent inhibitor of PC2, the most important function of 7B2 as regardsthis enzyme appears to be the absolute requirement of PC2 for 7B2 in thegeneration of active enzyme, recently corroborated through production of anull animal that lacks PC2 activity. The purpose of the present study wasto determine whether proSAAS exerts effects on PC1 other than inhibition,and to establish functional similarities and differences between 7B2 andproSAAS. We first asked whether the N-terminal domain of proSAAS(proSAAS-(1-180)) could stabilize PC1 activity, similar to the effect ofthe N-terminal domain of 7B2 on PC2. Recombinant His-taggedproSAAS-(1-180) had no effect on PC1 activity in vitro and was unable toprotect PC1 from thermal denaturation. Transient cotransfection ofproSAAS-(1-225) cDNA with PC1 cDNA into HEK 293 cells reduced the amountof PC1 activity detected in the medium. Surprisingly, cotransfection ofproSAAS-(1-180) cDNA, encoding a protein that lacks the inhibitoryC-terminal domain peptide, also reduced the activity of PC1 detected inthe medium, but the mass of PC1 secreted into the medium was increased,suggesting a proSAAS-mediated inactivation reaction. Similar results wereobserved in CHO/PC1 cells stably transfected with pro-SAAS-(1-180). Stabletransfection of SAAS cDNAs into AtT-20 cells was used to examine the roleof proSAAS in a neuroendocrine setting. Unlike 7B2, proSAAS-(1-225) wasable to slow convertase-mediated processing of proopiomelanocortin andproenkephalin; however, similarly to 7B2, proSAAS expression did notresult in any accumulated differences in the content of cellular processedpeptide. In summary, although both proSAAS and 7B2 potently inhibit PCenzymes via a C-terminal peptide, their intracellular interactions withPCs appear to differ significantly, with each binding protein exhibitingunique properties.

• Mishra AK, Mahale SD, Iyer KS
• Mapping the receptor binding regions of human chorionic gonadotropin (hCG)using disulfide peptides of its beta-subunit: possible involvement of thedisulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding ofthe hormone.
• J Pept Res. 2001; 58: 17-26
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• Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormoneessential for the establishment and maintenance of pregnancy. The alpha-and beta-subunits of hCG are highly cross-linked internally by disulfidebonds which seem to stabilize the tertiary structures required for thenoncovalent association of the subunits to generate hormonal activity. Thepurpose of this study was to delineate the role of the disulfide bonds ofhCGbeta in receptor binding of the hormone. Six disulfide peptidesincorporating each of the six disulfide bonds of hCGbeta were synthesizedand screened, along with their linear counterparts, for their ability tocompetitively inhibit the binding of [125I] hCG to sheep ovarian corporaluteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to beapproximately 4-fold more potent than the most active of its linearcounterparts in inhibiting radiolabeled hCG from binding to its receptor.Similarly, disulfide peptide Cys (23-72) exhibited receptor bindinginhibition activity, whereas the constituent linear peptides were found tobe inactive. The results suggest the involvement of the disulfide bondsCys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptorbinding of the hormone. This study is the first of its kind to usedisulfide peptides rather than linear peptides to map the receptor bindingregions of hCG.

• Palecanda A, Kobzik L
• Receptors for unopsonized particles: the role of alveolar macrophagescavenger receptors.
• Curr Mol Med. 2001; 1: 589-95
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• The lung is constantly exposed to potentially pathogenic particles andmicroorganisms. Alveolar macrophage (AM) binding of inhaled environmentalparticles is a critical first step in phagocytosis and clearance, and mustbe accomplished without the benefit of opsonization by specificantibodies. Opsonin-independent phagocytosis is initiated by directrecognition of phagocytic target. The identities of receptors on AMs thatmediate unopsonized particle binding were, until recently, not known.Using flow cytometry, monoclonal antibody and expression cloningtechniques we have found a major role for the scavenger receptor, MARCO inAM binding of particles and bacteria. In this review we will discuss therole of scavenger receptors in AM binding of unopsonized particles and theuse of flow cytomety in analyzing AM-particle interaction. We will alsodiscuss other non-scavenger receptors involved in opsonin-independentphagocytosis.

• Wang Y, Bernard MP, Moyle WR
• Bifunctional hCG analogs adopt different conformations in LH and FSHreceptor complexes.
• Mol Cell Endocrinol. 2000; 170: 67-77
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• Human reproduction requires specific interactions between follitropin(hFSH) and its receptor (FSHR) and between lutropin (hLH) orchoriogonadotropin (hCG) and the lutropin receptor (LHR). Substitution ofhFSH residues between hCG beta-subunit cysteines 11-12 creates abifunctional analog that binds both receptors. To understand the basis ofthis observation, we used antibody probes to compare the conformations ofbifunctional analogs before and after they were complexed with eachreceptor. Introduction of hFSH residues between cysteines 11-12 changed adistant conformation-sensitive region created by the juxtaposition of thesubunit aminotermini. This region, found not to contact either receptor,was altered further when bifunctional ligands bound FSHR. All othersurfaces, detected in LHR complexes, were also recognized in FSHRcomplexes, an indication that bifunctional ligands bind both receptors insimilar orientations. These observations suggest that unlike hCG or hFSH,bifunctional gonadotropins can acquire "lutropin" and "follitropin"conformations, a phenomenon accentuated by receptor contacts.

• Keri RA, Lozada KL, Abdul-Karim FW, Nadeau JH, Nilson JH
• Luteinizing hormone induction of ovarian tumors: oligogenic differencesbetween mouse strains dictates tumor disposition.
• Proc Natl Acad Sci U S A. 2000; 97: 383-7
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• The use of fertility drugs has continued to grow since their introductionin the 1960s. Accompanying this increase has been the speculation thatrepetitive use of these drugs can cause ovarian tumors or cancer. Werecently reported that transgenic mice with chronically elevatedluteinizing hormone (LH), an analog of which is commonly used in fertilityregimens, develop granulosa cell (GC) tumors. In this report we show thatLH induction of these tumors is highly dependent on genetic background. InCF-1 mice, chronically elevated LH invariably causes GC tumors by 5 monthsof age. However, in hybrid mice generated by crossing CF-1 males withC57BL/6, SJL, or CD-1 females, elevated levels of this same hormone causea completely different phenotype resembling a luteoma of pregnancy. Wealso show that three genes likely control these alternative hormonalresponses. This clinical correlate of elevated LH reveals remarkablydistinct, strain-dependent, ovarian phenotypes. In addition, these resultssupport the rare incidence of GC tumors in the human population, andsuggest that the ability of certain fertility drugs to cause ovariantumors may depend on an individual's genetic predisposition.

• Tegoni M, Spinelli S, Verhoeyen M, Davis P, Cambillau C
• Crystal structure of a ternary complex between human chorionicgonadotropin (hCG) and two Fv fragments specific for the alpha andbeta-subunits.
• J Mol Biol. 1999; 289: 1375-85
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• Human chorionic gonadotropin (hCG), is a placental hormone which exertsits major effect by stimulating progesterone production, cruciallysustaining the early weeks of pregnancy. Detection of hCG with specificmonoclonal antibodies (mAbs) has become the chosen means for pregnancydiagnosis. We have used antibody Fv fragments derived from twohigh-affinity mAbs, one against the alpha and the other against thebeta-hCG subunit to enable the crystallisation of intact or desialylatedhCG. Crystals of a ternary complex composed of Fv anti-alpha/hCG/Fvanti-beta were found to diffract to 3.5 A resolution, and the structurewas solved by molecular replacement. In the crystal, the two Fvs keep hCGas in a molecular cage, providing good protein-protein contacts andleaving enough space for the saccharides to be accommodated in the cellsolvent. The two Fvs were found not to interact directly through theircomplementary-determining regions with the hCG saccharides, but only withthe protein. The hCG structure in the ternary complex was very close tothat of the HF partially deglycosylated hormone, thus indicating thatneither the saccharides nor the Fvs had any substantial influence onhormone structure.

• Lin W, Ransom MX, Myers RV, Bernard MP, Moyle WR
• Addition of an N-terminal dimerization domain promotes assembly of hCGanalogs: implications for subunit combination and structure-functionanalysis.
• Mol Cell Endocrinol. 1999; 152: 91-8
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• Human chorionic gonadotropin (hCG) is a heterodimeric placentalglycoprotein hormone that acts through ovarian lutropin receptors (LHR) tomaintain early pregnancy. Its ability to distinguish LHR and follitropinreceptors (FSHR) is controlled by 20 beta-subunit 'seatbelt' residues thatsurround alpha-subunit loop 2. Positively charged amino acids betweenresidues 93-100, a small loop within the seatbelt, have been postulated tomake essential LH receptor contacts. Previous studies showed that analogscontaining negatively charged amino acids in this small loop had 5-10% theactivity of hCG and 1-10% the lutropin activities of hCG/hFSH chimericanalogs capable of binding LHR and FSHR. These effects might be due to theinfluence of these residues on receptor contacts or on hormoneconformation. During efforts to distinguish these possibilities, weincreased and decreased the number of residues in this loop, mutations weanticipated would distort its conformation. Consistent with thissupposition, these changes inhibited dimer formation, precludingassessment of these mutations on hormone activity. Addition of Fos and Jundimerization domains to the N-termini of hCGalpha- andhCG/hFSHbeta-subunit chimeras overcame the effects of the seatbeltmutations on subunit combination and enabled preparation of heterodimerscontaining six, seven, or nine residues in their seatbelt loops. These had0.1-10% the lutropin and 3-60% the follitropin activities of bifunctionalchimeras containing 8 residues derived from hCG in the seatbelt loop. Theabilities of N-terminal dimerization domains to promote subunitcombination may permit structure/function analysis of other residues thatinfluence heterodimer formation.

• Cosowsky L, Lin W, Han Y, Bernard MP, Campbell RK, Moyle WR
• Influence of subunit interactions on lutropin specificity. Implicationsfor studies of glycoprotein hormone function.
• J Biol Chem. 1997; 272: 3309-14
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• Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) areheterodimeric glycoprotein hormones required for reproduction. Both bindrat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs)1000-10,000 fold better than bLH. We tested the premise that thisdifference in affinity could be used to identify lutropin receptorcontacts. Heterodimers containing hCG/bLH alpha- or beta-subunit chimerasthat bound hLHR like hCG (or bLH) were expected to have hCG (or bLH)residues at the receptor contact sites. Analogs containing one subunitderived from hCG bound hLHR much more like hCG than bLH, indicating thateach bLH subunit contains all the residues sufficient for high affinityhLHR binding. Indeed, the presence of bovine alpha-subunit residuesincreased the activities of some hCG analogs. The low hLHR activity of bLHwas due primarily to an interaction between its alpha-subunit andbeta-subunit residue Leu95. Leu95 does not appear to contact the hLHRsince it did not influence the hLHR activity of heterodimers containinghuman alpha-subunit. These observations show that interactions within andbetween the subunits can significantly influence the activities oflutropins, thereby confounding efforts to identify ligand residues thatcontact these receptors.

• Dias JA
• Human follitropin heterodimerization and receptor binding structuralmotifs: identification and analysis by a combination of synthetic peptideand mutagenesis approaches.
• Mol Cell Endocrinol. 1996; 125: 45-54
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• The family of human glycoprotein hormones, including follitropin (FSH),are heterodimeric proteins, each composed of single alpha- andbeta-subunits that are tightly associated but non-covalently linked. Tostudy structure and function relationships of FSH, synthetic peptides wereused to inhibit subunit association, to map epitopes of FSH antibodies andas antigens to generate site specific antipeptide antibodies which couldbe used for topographic analysis. Interpretation of such results aregenerally more straightforward than when peptides are used withradioreceptor assays or in cell cultures which are complex systems. Thedata we collected using the synthetic peptide approach suggested that FSHresidues homologous to human chorionic gonadotropin (hCG) loops L3 betaand L2 alpha are involved in subunit contact. FSH residues homologous tohCG loops L2 beta and L3 alpha seemed involved in receptor binding. LoopL2 beta also seemed involved in subunit contact. Those data provided arationale for extensive mutagenesis of the four regions of hFSH.Mutagenesis data provided additional information and higher resolution offunction when combined with the three dimensional structure of hCG. In theaggregate, this information has provided a reasonable model of thereceptor binding site of hFSH. Our current model of the FSH receptor siteis that of a discontinuous functional epitope including L3 beta, L2 alphaand L3 alpha. The juxtaposition of residues beta D93, alpha K5 1, alphaY88 and of alpha Y89 in the 'binding-facet' of hFSH suggest thefeasibility of designing a synthetic peptide mimetic of FSH. Additionalresidues of the alpha-subunit are involved, along this facet of themolecule. The data collected studying hFSH therefore demonstrates that thealpha-subunit features prominently in the mechanism of FSH binding to andstabilizing the interaction with its receptor. In contrast, thebeta-subunit determinant loop serves as discriminator in addition tostabilizing the binding interaction whereas mutagenesis data indicatesthat L2 beta does neither. Instead, L2 beta appears to stabilize FSHconformation, possibly, the alpha-subunit, required for competent binding.In this regard, synthetic peptides provided data which were a useful guideto plan mutagenesis studies and which contributed to the process ofunderstanding the structure and function of the gonadotropins.

• Kisselev O, Pronin A, Ermolaeva M, Gautam N
• Receptor-G protein coupling is established by a potential conformationalswitch in the beta gamma complex.
• Proc Natl Acad Sci U S A. 1995; 92: 9102-6
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• Receptor-G protein interaction is characterized by cycles of associationand dissociation. We present evidence which indicates that duringreceptor-G protein interaction, the C-terminal tail of the G protein gammasubunit, which is masked in the beta gamma complex, is exposed andestablishes high-affinity contact with the receptor. This potentialconformational switch provides a mechanism to regulate receptor-G proteincoupling. This switch may also be significant for the role of the betagamma complex in regulation of effector function.

• Young PR
• Protein hormones and their receptors.
• Curr Opin Biotechnol. 1992; 3: 408-21
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• Technological advances in the isolation and characterization of novelreceptors have led to a significant increase in our understanding ofprotein-ligand binding to receptors and the means by which responses aretriggered. Hormones and their receptors are composed of structurallyconserved domains, and several ligands appear to use similar surfaceregions for receptor binding. A key event in signal transduction is theaggregation by the ligand of one or more receptor subunits, and this caninclude the sharing of subunits between different ligands. These findingshave allowed the design of ligands with receptor-antagonist properties.

• Jameson JL et al.
• Glycoprotein hormone alpha-subunit-producing pituitary adenomas in ratstreated for one year with calcitonin.
• Am J Pathol. 1992; 140: 75-84
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• Calcitonin, a calcium-lowering hormone, has been associated with anincreased incidence of nonfunctioning pituitary tumors in rats. In thisstudy, rats were treated with calcitonin (80 IU/kg/d) for 52 weeks. Aftertreatment with calcitonin, immunohistochemistry and in situ hybridizationanalyses demonstrated that most pituitary tumors expressed theglycoprotein hormone alpha-subunit. Expression of the alpha-subunit wasidentified rarely in hyperplastic lesions of control animals. Serum levelsof GH, PRL, ACTH, LH, and FSH were unchanged in calcitonin-treated ratsrelative to controls. However, TSH levels were increased 2.1 fold afterchronic treatment with calcitonin in both male and female rats (P lessthan 0.001). The level of glycoprotein hormone alpha-subunit was markedlyincreased (20-fold) in male rats with smaller elevations in female rats.Time course studies demonstrated that increases in serum alpha-subunitlevels could be detected by 24 weeks of treatment and that elevations inalpha-subunit were present in the majority of animals by 40 weeks oftreatment with calcitonin. The authors conclude that high doses ofcalcitonin, administered to rats for 6 months or longer, increases theincidence of alpha-subunit-producing pituitary tumors.

• Halberg DF, Proulx G, Doege K, Yamada Y, Drickamer K
• A segment of the cartilage proteoglycan core protein has lectin-likeactivity.
• J Biol Chem. 1988; 263: 9486-90
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• A segment of 130 residues near the COOH terminus of the proteoglycan coreprotein derived from rat cartilage is highly homologous to thecarbohydrate-recognition domain of the chicken hepatic lectin and othervertebrate carbohydrate-binding proteins. This portion of the protein hasbeen expressed in an in vitro transcription and translation system and hasbeen tested for its ability to interact with carbohydrates using affinitychromatography on immobilized sugars. A distinct specificity of thebinding interaction is demonstrable, with fucose and galactose being thepreferred ligands. However, the affinity of the expressed domain of theproteoglycan core protein is lower than that of the other known bindingdomains, since it elutes from the columns even in the presence of Ca2+.

• Sheary CB
• Can orthodontia influence pituitary gland function and learning ability?
• Basal Facts. 1986; 8: 71-4

• Baenziger JU
• The role of glycosylation in protein recognition. Warner-LambertParke-Davis Award lecture.
• Am J Pathol. 1985; 121: 382-91

• Roser JF, Papkoff H, Murthy HM, Chang YS, Chloupek RC, Potes JA
• Chemical, biological and immunological properties of pituitarygonadotropins from the donkey (Equus asinus): comparison with the horse(Equus caballus).
• Biol Reprod. 1984; 30: 1253-62
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• Donkey gonadotropins (donkey luteinizing hormone, dLH; donkeyfollicle-stimulating hormone, dFSH) have been isolated in purified formfrom 191 donkey pituitaries using essentially the same procedurespreviously employed for the purification of equine gonadotropins.Chemically, dLH and dFSH were observed to be similar to equine LH (eLH)and FSH (eFSH) in fractionation behavior and glycoprotein nature. Twoforms of the dFSH molecule were observed, as is the case for eFSH. DonkeyLH had significantly less total carbohydrate (13.5%) and sialic acid(1.9%) than eLH (26.7% and 5.8%, respectively). Carbohydrate (17-21%) andsialic acid (2.4%) content of the two dFSH preparations closely resembledthat of eFSH. A slightly higher tyrosine content in the donkeygonadotropins was noted in a comparison of amino acid compositions.Immunologically, in a heterologous FSH radioimmunoassay (RIA), dFSHpreparations were equal to or twice as active as eFSH preparations.However, in homologous RIAs for equine chorionic gonadotropin (eCG), eFSHand eLH, both the dLH and dFSH preparations were considerably less activethan the equine gonadotropins, and their inhibition curves were allnonparallel. Biologically, in the Steelman-Pohley assay both dFSHpreparations were equipotent and as potent as eFSH (approximately 40 timesNIH-FSH-S12). In the Sertoli cell assay for cAMP (FSH assay) and theLeydig cell assay for testosterone (LH assay), both dFSH and dLH were 2-or 6-fold more active than eFSH and eLH, respectively. In rat and equinetestis FSH homologous radioreceptor assays, dFSH preparations were asactive and up to 6-fold more active than eFSH. In contrast, dLH was10-fold less active than eLH in the equine LH homologous radioreceptorassay. Unlike eLH, dLH was found to possess little intrinsic FSH activityor FSH inhibitory activity, and the small amount of FSH activity observedwas most likely due to FSH contamination. Therefore, eLH behaves much likeeCG (pregnant mare's serum gonadotropin, PMSG) which also possesses bothLH and FSH activity. In contrast, dLH behaves more like donkey chorionicgonadotropin (dCG) which possesses only a low degree of FSH activity.

• Hwang J, Menon KM
• Spatial relationships of the human chorionic gonadotropin (hCG) subunitsin the assembly of the hCG-receptor complex in the luteinized rat ovary.
• Proc Natl Acad Sci U S A. 1984; 81: 4667-71
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• In an attempt to examine the spatial relationships of the human chorionicgonadotropin (hCG) subunits in the assembly of the hCG-receptor complex,the recombined 125I-labeled hCG, with label in either the alpha subunit orthe beta subunit, was cross-linked to the luteinizing hormone (LH)/hCGreceptor. The efficacy of the cross-linking of the 125I-alpha subunit orthe 125I-beta subunit of hCG to the LH/hCG receptor was then examined. Theautoradiographic profile of 125I-hCG-receptor complex containing the labelin the alpha subunit of hCG showed that the alpha subunit can cross-linkwith all four subunits of the LH/hCG receptor. However, only one faintlabeled band, corresponding to Mr = 68,000, was detected when the125I-hCG-receptor complex with label in the beta subunit was subjected tosodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducingconditions. When the electrophoresis was performed under nonreducingconditions, the Mr 68,000 band disappeared concomitantly with theaccumulation of radioactivity in the high molecular weight region. Theseresults indicated that the beta subunit of hCG, unlike the alpha subunit,can cross-link only weakly with the smallest subunit of the LH/hCGreceptor. A comparison of the differential effectiveness of thecross-linking of 125I-alpha subunit with 125I-beta subunit of hCG to theLH/hCG receptor suggests that both alpha and beta subunits of hCG areintimately associated with the receptor, but the bulk of the beta subunitof hCG is buried in between the receptor and the alpha subunit of hCG. Onthe basis of our data, a model for the spatial arrangement of hCG subunitsin the hCG-receptor complex is proposed.

• Chin WW, Godine JE, Klein DR, Chang AS, Tan LK, Habener JF
• Nucleotide sequence of the cDNA encoding the precursor of the beta subunitof rat lutropin.
• Proc Natl Acad Sci U S A. 1983; 80: 4649-53
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• We have determined the nucleotide sequences of cDNAs encoding theprecursor of the beta subunit of rat lutropin, a polypeptide hormone thatregulates gonadal function, including the development of gametes and theproduction of steroid sex hormones. The cDNAs were prepared from poly(A)+RNA derived from the pituitary glands of rats 4 weeks after ovariectomyand were cloned in bacterial plasmids. Bacterial colonies containingtransfected plasmids were screened by hybridization with a 32P-labeledcDNA encoding the beta subunit of human chorionic gonadotropin, a proteinthat is related in structure to lutropin. Several recombinant plasmidswere detected that by nucleotide sequence analyses contained codingsequences for the precursor of the beta subunit of lutropin. Completedetermination of the nucleotide sequences of these cDNAs, as well as ofcDNA reverse-transcribed from pituitary poly(A)+ RNA by using a syntheticpentadecanucleotide as a primer of RNA, provided the entire 141-codonsequence of the precursor of the beta subunit of rat lutropin. Theprecursor consists of a 20 amino acid leader (signal) peptide and anapoprotein of 121 amino acids. The amino acid sequence of the rat lutropinbeta subunit shows similarity to the beta subunits of the ovine/bovine,porcine, and human lutropins (81, 86, and 74% of amino acids identical,respectively). Blot hybridization of pituitary RNAs separated byelectrophoresis on agarose gels showed that the mRNA encoding the lutropinbeta subunit consists of approximately 700 bases. The availability ofcDNAs for both the alpha and beta subunits of lutropin will facilitatestudies of the regulation of lutropin expression.

• Goodwin RG, Moncman CL, Rottman FM, Nilson JH
• Characterization and nucleotide sequence of the gene for the common alphasubunit of the bovine pituitary glycoprotein hormones.
• Nucleic Acids Res. 1983; 11: 6873-82
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• The gene coding for the common alpha subunit of the bovine pituitaryglycoprotein hormones was isolated from a bovine genomic library. The genespans roughly 16.5 kbp, contains three intervening sequences, and codesfor a message of approximately 730 nucleotides. The complete coding regionof the gene was sequenced as well as 315 nucleotides of 5' flankingsequence and the entire intron C. Only a single base difference was foundwhen the sequence of the gene was compared with that of the cDNA. Genomicblotting experiments suggest the presence of a single alpha subunit gene.Comparison of the bovine and human alpha subunit genes indicated that thehigh level of homology observed in the coding regions has been maintainedthroughout the 5' and 3' untranslated regions, and at least 90 nucleotidesof the 5'flanking regions. Additionally, there is an 18 base pair sequencepresent in both the 5' flanking and 5' untranslated regions of the genethat is homologous to a region of the chick ovalbumin gene. This ovalbuminsequence has been suggested as a binding site for the progesteronereceptor-complex.

• Korczyn A
• [Hypophyseal hormones--neurotransmitters?].
• Harefuah. 1980; 99: 441-2

• Domagk GF
• [Protein stabilization by means of covalent binding with carbohydrates].
• Hippokrates. 1978; 49: 264-5

• Cuibus F, Vladescu C, Vascan C, Maniceanu E
• [Hormonal regulation of glycemia in the aged].
• Fiziol Norm Patol. 1972; 18: 451-9