Gonadotropins are heterodimeric glycoprotein hormones that control vertebrate fertility through their actions on gonadal lutropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH) receptors. The beta-subunits of these hormones control receptor binding specificity; however, the region of the beta-subunit that contacts the receptor has not been identified. By a process of elimination we show this contact to be the portions of beta-subunit loops one and three found in a hormone groove created by the juxtaposition of the alpha- and beta-subunits. Most other regions of the beta-subunit can be recognized by antibodies that bind to human chorionic hormone (hCG)-receptor complexes or replaced without disrupting hormone function. Using a series of bovine LH/hCG and human FSH/hCG beta-subunit chimeras we identified key hCG beta-subunit residues in the epitopes of two antibodies that bind to hCG-receptor complexes. These epitopes include the surfaces of beta-subunit loops one and three near residue 74 on the outside of the hormone groove and parts of the C-terminal end of the "seat belt" that holds the two subunits together. The antibody that recognized residue 74 bound to receptor complexes containing most mammalian lutropins better than to the free hormones, an indication that the outside surface of the beta-subunit groove is altered during hormone binding. This region of the beta-subunit is furthest from the alpha-subunit and is recognized equally well in the free beta-subunit and in the heterodimer. Thus, the receptor associated increase in antibody binding appears due to an interaction of this portion of the beta-subunit with the receptor and not to an effect of the receptor on the relative positions of the alpha- and beta-subunits. Unlike most previous studies designed to identify portions of the beta-subunit likely to contact the LH receptor, this indirect approach provides data that are more easily interpreted because it does not rely on the use of mutations that disrupt hormone function. The approach described here should be valuable for studying the receptor interactions of other complex ligands.

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000 -10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH ␣-or ␤-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine ␣-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its ␣-subunit and ␤-subunit residue Leu 95 . Leu 95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human ␣-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.The gonadotropins human lutropin (hLH), 1 human chorionic gonadotropin (hCG), and human follitropin (hFSH) are essential for reproduction and have been used for many years to enhance human fertility. Development of clinically useful agonist and antagonist analogs would be facilitated by knowledge of how these ligands interacted with their receptors. Two radically different models of gonadotropin-receptor interaction have been proposed (1, 2) based on the crystal structures of hCG (3, 4) and ribonuclease inhibitor (5, 6), a protein containing a leucine-rich repeat motif thought to be similar to those in the glycoprotein hormone receptors. These models could be readily distinguished if the portions of the hormone that contacted their receptors were known.Like other glycoprotein hormones, the gonadotropins are heterodimers that contain a conserved ␣-subunit and a hormone-specific ␤-subunit (7). Each subunit is divided into three large loops by a cysteine knot (3, 4), and the heterodimer is stabilized by a portion of the ␤-subunit termed the "seat belt" (3) that is wrapped around ␣-subunit loop 2. Based on the activities of chemically and enzymatically modified hormones (summarized by Pierce and Parsons (7)), synthetic hormone fragments (8 -12), and analogs prepared by site-directed mutagenesis (13-19), residues throughout both hormone subunits have been suggested to participate in essential high affinity hormone receptor contacts. Surprisingly, some hCG residues proposed to contact LH receptors are in regions recognized by monoclonal antibodies that bind to hCG-receptor complexes (1,20). Others thought to be essential for receptor contacts can be replaced without disrupting receptor binding. For example, replacing hCG seat belt residues 101-109 with their hFSH counterparts ...

Summary The feasibility of obtaining clonal lines with replicating, multicopy geminivirus vectors by direct DNA trans‐formation of cultured tobacco cells was studied. The replicating vectors pTGA32 and pST31 are based on the tomato golden mosaic virus (TGMV) A genome and encode the neomycin phosphotransferase type II (NPT‐II) enzyme that confers kanamycin resistance to plant cells. Following introduction into plant cells, unit‐length viral genomes were released from the tandem repeats and replicated. In protoplasts, replication of unit‐length pTGA32 and pST31 was about as efficient as replication of unit‐length DNA A from plasmid pTGA26, which contains 1.5 copies of wild‐type DNA A. Tobacco suspension culture cells were bombarded with the recombinant DNA A constructs and selected for kanamycin resistance. The number of kanamycin‐resistant clones per bombardment was about the same when the TGMV DNA A vectors or a non‐replicating plasmid (pLC14) which also encodes NPT‐II was used. Replicating, unit‐length DNA A in up to approximately 1000 copies per cell was found in about 10% of the kanamycin‐resistant clones selected following bombardment of cells with TGMV vectors. The results suggest that geminiviruses may serve as useful multicopy vectors in cultured cells.

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The actions of prostaglandins (PG) on cAMP in dispersed chief cells from guinea pig stomach were examined and compared to the actions of these agents on pepsinogen secretion. Maximal concentrations of A, B, or E prostaglandins caused a 2-5-fold increase in pepsinogen secretion and cellular cAMP. The relative order of potency for these actions was PGEs greater than PGAs greater than PGBs. Detection of prostaglandin-induced changes in cAMP was enhanced by adding a phosphodiesterase inhibitor to the incubation solution. The time courses for the effects of prostaglandins on pepsinogen secretion and cAMP were similar. With PGE1 an increase in cAMP and pepsinogen secretion was detected by 1 min and was maximal by 7.5 min. Although significant increases in cAMP were detected with a ten-fold lower concentration of PGEs than PGAs, a maximal increase in cAMP was observed with the same concentration, 30 microM, of either agent. These data indicate that prostaglandins that stimulate pepsinogen secretion increase cAMP in dispersed chief cells. However, comparison of the dose-response curves for the actions of prostaglandins on pepsinogen secretion and cAMP revealed that detectable increases in cAMP occurred with concentrations of these agents that were about ten-fold greater than those needed to stimulate pepsinogen secretion. Therefore, although the similarity in the kinetics and relative potencies of prostaglandin-induced changes in cAMP and enzyme secretion provides further evidence that changes in cAMP play a role in the mediation of prostaglandin-induced pepsinogen secretion, the present data suggest the involvement of a cellular messenger in addition to cAMP.

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