The luteinizing hormone/chorionic gonadotropin receptor is a member of the seven-transmembrane receptor family. It is coupled, presumably via G s and G q , to two signal pathways involving adenylyl cyclase/cAMP and phospholipase C/inositol phosphate (IP). Little is known about the events prior to G-protein coupling: for example, whether these signals are generated from a single or multiple independent origins and mechanisms, when and where they diverge, and how they are transduced. We report novel observations that the cAMP signal and the IP signal originate and diverge upstream of G-protein coupling. The LH/CG 1 receptor is a member of the seven-transmembrane (TM) receptor family and comprises two distinct functional domains consisting of the extracellular N-terminal half and the membrane-associated C-terminal half. These two domains are encoded by 11 exons (1, 2). Exons 1-10 encode the extracellular N-terminal domain, which alone is responsible for high affinity hormone binding (3)(4)(5). This distinguishes the LH/CG receptor and other glycoprotein hormone receptors from all other seven-TM receptors, which have the high affinity ligand binding sites in the membrane-associated domain (6). Exon 11 of the LH/CG receptor encodes the membrane-associated domain, which is capable of low affinity hormone binding and receptor activation (5, 7). This membrane-associated domain includes seven-TM helices joined together by three exoloops and three cytoloops.The LH/CG receptor is coupled to the adenylyl cyclase and PLC signal pathways, inducing the production of cAMP and inositol phosphates (IPs) as intracellular signal molecules (8). These signal molecules are produced when the receptor couples, presumably through G s and G q (9), to adenylyl cyclase and PLC, respectively (10). However, little is known about the events prior to G-protein coupling (6) (in particular, when more than one signal is involved). As a first step to lead discussion, several simple mechanisms are hypothesized for dual signals as shown in Fig. 1. Hormonal signal generation and transfer can be likened to an electrical circuit. Contact between the hormone and the receptor acts as an on/off signal switch and the receptor conducts the signal to the terminal cytodomain G-protein dock. Signals may be generated and transferred by single switch/single conductor, single switch/dual conductors, or dual switches/dual conductors. We will examine these models in light of the existing evidence.Recent investigations have identified several amino acids of the LH/CG receptor that are important for receptor activation (11)(12)(13)(14)(15)(16)(17)(18)(19). Most of these amino acids are present in cytoplasmic or TM domains. Only a few were found in the extracellular regions of the LH/CG receptor (11,12,(17)(18)(19). The added significance of extracellular amino acids in receptor activation is that they are near or at the hormone binding domain and could be involved in signal generation (6). Although their involvement in cAMP induction has been extensively examined, non...

The human follicle-stimulating hormone receptor (FSH-R) consists of two distinct domains of >330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain. The exodomain alone binds hormone with high affinity, whereas the endodomain is the site of receptor activation. Coordination of these two domains is essential for successful hormone action but little is known about their functional and structural relationship. In this communication, we report that exoloop 3 of FSH-R constrains follicle-stimulating hormone binding to the exodomain. When the FSH-R exodomain was prepared by truncating its endodomain, the hormone binding affinity of the exodomain was slightly improved, compared with the wild type receptor. The binding affinity was further improved by >3-fold when the exodomain was attached to the membrane-associated domain of CD8. These results suggest that the FSH-R endodomain attenuates hormone binding at the exodomain. As a first step to test this hypothesis, the 11 amino acids except Ala 589 of exoloop 3 were individually substituted with Ala. Ala substitution for Leu 583 or Ile 584 improved the hormone binding affinity by 4 -6-fold while totally abolishing cAMP induction, indicating an inverse relationship. The Ala substitution for Lys 580 or Pro 582 had a similar trend but to a lesser extent. This significant improvement in the binding affinity suggests that the four residues at the N-terminal region of exoloop 3 interact with the exodomain and constrain the hormone binding in the wild type receptor. This effect is specific since substitutions for other than the 4 residues did not improve the hormone binding affinity. Computer modeling shows that the 4 residues can be positioned on one side of exoloop 3. This result and the apparent inverse relationship of hormone binding and cAMP induction suggest that these two essential functions may work against each other. Therefore, hormone binding might be compromised to preserve cAMP inducibility while maintaining a reasonably high, but below maximum, binding affinity. The FSH1 receptor and other glycoprotein hormone (LH/CG and TSH) receptors belong to a structurally unique subfamily of G protein-coupled receptors (1). Unlike other receptor subfamilies, they comprise two equal halves, an extracellular Nterminal half (exodomain) and a membrane-associated C-terminal half (endodomain) (2-5) as shown in Fig. 1. The exodomain is ϳ350 amino acids long, which alone is capable of high affinity hormone binding (6 -9) with hormone selectivity (10 -12) but without hormone action (8, 13). Receptor activation occurs in the endodomain (14), which is structurally equivalent to the entire molecule of many other G protein-coupled receptors (15). Existing evidence suggests that glycoprotein hormones initially bind to the exodomain, and then the resulting hormone-exodomain complex interacts with the endodomain (1). This secondary interaction is responsible for signal generation (1, 14). Despite the importance of this secondary contact and the relationship...

The luteinizing hormone/choriogonadotropin (CG) receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. It is comprised of an extracellular N-terminal half of 341 amino acids and a membrane-associated C-terminal half of 303 amino acids. The N-terminal half is capable of high affinity hormone binding whereas the C-terminal half is capable of low affinity hormone binding and receptor activation. However, the precise location of the receptor activation site is currently unknown. We present evidence for the first time that Lys583 of exoloop 3 is crucial and irreplaceable for receptor activation to induce cAMP synthesis. Exoloop 3 is comprised of 11 amino acids and flanked by two Lys residues, Lys573 and Lys583, that are located at the boundaries with the transmembrane columns 6 and 7, respectively. All substitutions including Arg for Lys583 did not affect the high affinity human CG binding, but they resulted in the complete loss of cAMP synthesis induced by human CG. Ala substitutions of the other amino acids in exoloop 3 did not make such a dramatic impact on cAMP induction. The Ala scan revealed two distinct groups of amino acids in terms of their importance in cAMP induction, one group being more important than the other. Interestingly, these two groups of amino acids are arranged in an alternate sequence. This result suggests a specific structure similar to a beta-like structure for exoloop 3.

Trophoblastic neoplasms and choriocarcinoma cells express high levels of the hCG receptor. The hCG receptor is encoded by a single gene in chromosome 2p21-p16, spanning over -70 kb with 11 exons and 10 introns. Multiple mRNA species are produced from the gene utilizing two proximal promoters and several Sp-1 elements as well as proximal and distal suppressors. In fact, regulatory proteins which bind to one of these suppressors are expressed less in choriocarcinoma cell lines than in placenta. The LH/CG receptor is comprised of two structurally and functionally distinct domains, extracellular N-terminal exodomain and membrane embedded endodomain. These two domains can separately be expressed and processed, including folding. The exodomain alone has the high affinity hormone binding site but is not capable of generating hormonal signal. In contrast, the endodomain alone has the site for receptor activation. These two domains contact each other in holo-receptor and split receptor. This interaction, particularly through exoloops 2 and 3, constrains the high affinity hormone binding at the exodomain. Conversely, the exodomain could be involved in receptor activation. Therefore, these two domains are not entirely independent although they can be independently synthesized and processed. The existing evidence indicate that hCG and the receptor undergo multiple stages of interactions leading to receptor activation. Initial high affinity binding of hCG to the exodomain results into conformational adjustments of the hCG/exodomain complex. This leads to the secondary, low affinity contact of the hCG/exodomain complex with the endodomain. This secondary contact is responsible for generating signals. They are transduced through TM to the cytoplasmic portion (cytoloops and the C-terminal tail) of the receptor and then, transferred to cytoplasmic signaling molecules, such as G protein. Mutations in the exodomain and endodomain (N-extension, exoloops, TM, cytoloops, and cytoplasmic tail) have the potential to interfere with receptor activation at different steps, signal generation, transduction and transfer. Binding of hCG to the LH/CG receptor are known to induce two signals, one for adenylyl cyclase/ cAMP and the other for phospholipase C/inositol phosphate/diacylglycerol. The cAMP signal and IP signal diverge at the surface of the receptor. These independent signals are separately transduced through the transmembrane domains to the cytoplasmic part of the receptor, indicating the existence of the distinct transducers for each of the signals. Furthermore, it is likely that the divergent signals are separately transferred to cytoplasmic signal molecules such as G protein. In addition, each of the cAMP signal and IP signal consists of at least three separate subsignals: affinity signal, maximal production (efficacy) signal and basal level signal. In heterodimeric hCG, there are distinct parts responsible for high affinity receptor binding and receptor activation. Particularly, the C-terminal reduces of the alpha subunit play a cr...

The greatest challenge facing the U.S. Department of Energy is the remediation of the 1 x l0 s gal of high-level and low-level radioactive waste in the underground storage tanks (USTs) at its Hanford, Savannah River, Oak Ridge, Idaho, and Fernald sites. With current technologies, this remediation will cost at least 100 billion dollars. In an effort to reduce costs, improve safety, and minimize delays, the Underground Storage Tank-Integrated Demonstration was created for demonstration, testing, and evaluation (DT&E) of promising new technologies that can be used l i r for UST remediation. These demonstrations, which are typically at the pilot-plant scale, will determine which processes will be used in the full-scale remediation of the USTs. These DT&E studies are performed by the Characterization and Waste Retrieval Program or by the Waste Processing and Disposal Program (WPDP). This paper presents the technical progress and future plans of the WPDP projects. The 11 WPDP programs in FY 1993 focused on three problem areas, which involve the treatment of supernate, the treatment of sludge, and nitrate destruction and subsequent waste forms. The three supernate projects were primarily concerned with the development of the compact processing units. The four sludge studies were designed to develop a complete system-level plan for handling sludge and supernate, with particular emphasis on sludge-dissolution treatment, and to evaluate TRUEX and diamide solvent extraction processes for transuranic waste streams. The four projects on nitrate destruction and subsequent waste forms approached these problems in unique ways. In addition, a planned Request for Expression of Interest on organic destruction techniques from private industries and universities and the WPDP's future direction and programmatic issues are discussed.