Prema Narayan

Protein Engineering of a Novel Constitutively Active Hormone-Receptor Complex

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone consisting of an α and a β subunit that stimulates intracellular levels of cAMP via a G protein-coupled receptor. Herein we report the engineering and characterization of a novel molecule in which the receptor and its heterodimeric ligand were covalently linked in a single polypeptide chain. The hormone-receptor complex was expressed in cells transfected with this construct, but the cells were unable to bind significant amounts of exogenous hCG. However, cleavage of the hormone with a site-specific protease rendered the receptor accessible to exogenously added hormone. Cells transfected with the hCG-receptor construct contained elevated basal levels of cAMP; moreover, addition of hormone had no significant effect. These results are consistent with a strong and stable interaction between the single-chain hormone and its covalently linked receptor that results in a constitutively active complex.

hCG-receptor binding and transmembrane signaling

The technique of site-directed mutagenesis has proven to be quite powerful in elucidating contact sites involved in the interaction of the heterodimeric glycoprotein hormones and their respective seven transmembrane (TM) G protein-coupled receptors. Our laboratory has focused on identification of the minimum core sequences of the alpha and beta subunits required for bioactivity, the minimum length of a conjoined (yoked) single-chain hCG, the amino acid residues on hCG and the LH/CG-receptor (LH/CG-R) responsible for high-affinity binding, and the regions of the receptor that are involved in TM signaling. A number of amino acid residues have been mapped on the alpha and beta subunits of hCG that appear important in receptor binding. When projected onto the crystal structure of HF-treated hCG, these residues, by and large, cluster on one side of the molecule and cover a sizeable surface area, indicating that the hormone-receptor binding interface is rather extensive. Based on mutagenesis studies of several conserved ionizable amino acid residues in the extracellular domain (ECD) of LH/CG-R and a model that we, in collaboration with Drs Lapthorn and Isaacs, have developed for this region based on the crystal structure of porcine ribonuclease inhibitor, a charged region that appears to play an important role in hormone-receptor recognition has been identified. We have also delineated several regions of LH/CG-R that do not appear to participate in hCG binding but are involved in hCG-mediated signaling. These regions are located in the ECD and extracellular loop III just prior to entry into the membrane via TM helices I and VII, respectively, and in TM helices VI and VII. Similarly, a homologous region in the ECD of the FSH receptor, located with ten residues of TM helix I, is important in signaling but not hormone binding. These results suggest that ligand binding and ligand-mediated receptor activation are quasi-distinct, albeit sequential phenomena. Collectively, our mutagenesis and modeling studies, coupled with results from other laboratories, argue for a ligand-induced conformational change of the receptor that may involve a relative reorientation of the TM helices.

Structure‐Function Relationships of the Luteinizing Hormone Receptor

Abstract: Of the 800‐900 genes in the human genome that appear to encode G‐protein‐coupled receptors (GPCRs), two are known to encode receptors that bind the three heterodimeric human gonadotropins, luteinizing hormone (LH), chorionic gonadotropin (CG), and follicle‐stimulating hormone (FSH). LH and CG bind to a common receptor, LHR, and FSH binds to a paralogous receptor. These GPCRs contain a relatively large ectodomain (ECD), responsible for high‐affinity ligand binding, and a transmembrane portion, as in the other GPCRs. The ECD contains nine leucine‐rich repeats capped by N‐terminal and C‐terminal cysteine‐rich regions. The overall goal of this research is to elucidate the molecular mechanisms by which CG and LH bind to and activate LHR and the latter, in turn, activates Gsα. A combination of molecular modeling and site‐directed mutagenesis, coupled with binding and signaling studies in transiently transfected HEK 293 cells expressing wild‐type and mutant forms of LHR, has been used to develop and test models for the LHR ECD, the CG‐LHR ECD complex, and the structural changes in the transmembrane helices and intracellular loops, particularly loop 2, that accompany receptor activation. In addition, a single‐chain CG‐LHR complex was designed in which a fusion protein of the two subunits of human CG was linked to full‐length LHR. This ligand‐receptor complex was shown to be constitutively active in cellular models and in transgenic mice, the latter of which exhibit precocious puberty. From a combination of molecular modeling, site‐directed mutagenesis, genetic/protein engineering, and receptor characterization in cellular and animal models, considerable insight is being developed on the mechanisms of normal and aberrant activation of LHR.

Identification of Ionizable Amino Acid Residues on the Extracellular Domain of the Lutropin Receptor Involved in Ligand Binding

The LH receptor (LHR) is a G protein-coupled receptor characterized by a relatively large N-terminal extracellular domain responsible for high affinity ligand binding. Based on a model proposed for a major portion of the extracellular domain that contains a number of leucine-rich repeats, nine ionizable amino acid residues (Glu57, Glu80, Lys158, Glu181, Lys183, Glu184, Glu188, Lys190, and Asp206) were selected for charge reversal mutagenesis based on their locations in the proposed model and their potential to serve as ligand contact sites. Mutant LHR complementary DNAs were transiently transfected into COS-7 cells, and the expressed receptors were characterized by Western blot analysis, competitive ligand (hCG) binding, and ligand-mediated cAMP production. The most interesting mutants were K158E, K183E, E184K, and D206K, which were present on the plasma membrane fraction, as judged by Western blots, but were incapable of binding hCG and, of course, were deficient in hCG-mediated cAMP production. Other re...

Precocious Puberty and Leydig Cell Hyperplasia in Male Mice With a Gain of Function Mutation in the LH Receptor Gene

The LH receptor (LHR) is critical for steroidogenesis and gametogenesis. Its essential role is underscored by the developmental and reproductive abnormalities that occur due to genetic mutations identified in the human LHR. In males, activating mutations are associated with precocious puberty and Leydig cell hyperplasia. To generate a mouse model for the human disease, we have introduced an aspartic acid to glycine mutation in amino acid residue 582 (D582G) of the mouse LHR gene corresponding to the most common D578G mutation found in boys with familial male-limited precocious puberty (FMPP). In transfected cells, mouse D582G mLHR exhibited constitutive activity with a 23-fold increase in basal cAMP levels compared with the wild-type receptor. A temporal study of male mice from 7 days to 24 weeks indicated that the knock-in mice with the mutated receptor (KiLHR(D582G)) exhibited precocious puberty with elevated testosterone levels as early as 7 days of age and through adulthood. Leydig cell-specific genes encoding LHR and several steroidogenic enzymes were up-regulated in KiLHR(D582G) testis. Leydig cell hyperplasia was detected at all ages, whereas Sertoli and germ cell development appeared normal. A novel finding from our studies, not previously reported in the FMPP cases, is that extensive hyperplasia is commonly found around the periphery of the testis. We further demonstrate that the hyperplasia is due to premature proliferation and precocious differentiation of adult Leydig cells in the KiLHR(D582G) testis. The KiLHR(D582G) mice provide a mouse model for FMPP, and we suggest that it is a useful model for studying pathologies associated with altered LHR signaling.

A biologically active single chain human chorionic gonadotropin analog with altered receptor binding properties.

hCG is a heterodimer consisting of an alpha-subunit common among all members of the glycoprotein hormone family, LH, FSH, and TSH, and a unique beta-subunit responsible for receptor specificity. Biologically active single chain analogs of these hormones have been engineered in which the C-terminus of the beta-subunit was fused to the N-terminus of the alpha-subunit (N-beta-alpha-C) either with or without a linker such as the hCGbeta C-terminal peptide (CTP). This tandem order of subunits was chosen based on studies suggesting that the N-terminal region of hCGbeta and particularly the C-terminal region of the alpha-subunit are important in receptor binding and activation. Single chain hCG (YhCG1) can, in turn, be fused to the LH receptor to yield a hormone-receptor complex that is biologically active in transfected cells. Herein, we report the construction of a new single chain hCG analog (YhCG3) in which the C-terminus of the alpha-subunit is fused to the N-terminus of hCGbeta via a CTP (N-alpha-CTP-beta-C). Compared with YhCG1, this analog binds receptor with a 25- to 30-fold lower affinity, but, surprisingly, is capable of stimulating intracellular cAMP levels to the same extent. Furthermore, YhCG3 can be covalently linked to its receptor to produce a biologically active complex that results in elevated levels of basal cAMP in transfected cells. These results suggest that free N- and C-termini of hCGbeta and the alpha-subunit, respectively, are not essential for receptor binding and activation and that YhCG3 is in a more efficacious conformation for receptor activation than YhCG1.

Constitutively active luteinizing hormone receptors: Consequences of in vivo expression

Activating mutations in the luteinizing hormone receptor (LHR) gene are one of the most common mutations found in the gonadotropin receptor genes. Human males with these mutations exhibit precocious puberty while females do not have an obvious phenotype. To better understand the pathophysiology of premature LHR activation, transgenic mice have been generated with an activating mutation in LHR and a genetically engineered ligand-activated LHR. This review will summarize the major findings obtained with these two genetically modified mouse models and briefly discuss the similarities and differences between them and with the human phenotype.

Expression of functional lutropin/choriogonadotropin receptor in the baculovirus system

The LH/CG receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large glycosylated extracellular domain. The complete 674 amino acid residue rat receptor was expressed in Sf9 insect cells using the baculovirus expression system. Optimal expression under the control of the polyhedrin promoter was obtained at 72 h post-infection and a multiplicity of infection of 0.1. The recombinant LH/CG-R was expressed on the cell surface (ca. 4500 receptors/cell) and exhibited saturable, high affinity binding of human CG (hCG) with a Kd of 0.4 nM. There was no evidence of intracellular trapping of the receptor. The intracellular concentration of cAMP was increased in response to hCG binding. In contrast, baculovirus-expressed recombinant hCG only weakly stimulated intracellular cAMP levels at relatively high doses. Two forms of the receptor (approximately 75 and approximately 200 kDa) were detected by Western blot analysis. These results demonstrate that the full length LH/CG-R expressed in insect cells is functional in that it binds hormone with high affinity and is able to couple to adenylate cylase.

The Gonadotropin Hormones and Their Receptors

Abstract The gonads (ovary, testis) produce gametes (oocytes and spermatozoa). The pituitary glycoprotein hormones luteinizing hormone (LH, lutropin) and follicle-stimulating hormone (FSH, follitropin) are gonadotropins because their targets are the gonads. Another hormone produced by the syncytiotrophoblast of the human placenta is human chorionic gonadotropin (hCG, choriogonadotropin), and its activity is like LH. This review describes the protein structure, gene structure, and regulation of biosynthesis of both the gonadotropins and their membrane bound receptors. The functions of both hormones and receptors are covered extensively, as well as naturally occurring mutations that have been identified in disease states and therapies derived from their study.

Impact of a Constitutively Active Luteinizing Hormone Receptor on Testicular Gene Expression and Postnatal Leydig Cell Development

The actions of luteinizing hormone (LH) mediated through its receptor (LHR) are critical for testicular steroidogenesis and Leydig cell differentiation. We have previously characterized transgenic mice expressing a genetically engineered, constitutively active yoked hormone-receptor complex (YHR), in which a fusion protein of human chorionic gonadotropin (hCG) was covalently linked to LHR. Elevated testosterone levels were detected in male mice expressing YHR (YHR(+)) at 3 and 5 weeks of age, accompanied by decreases in testicular weight and serum levels of LH and follicle stimulating hormone (FSH). Here we report a temporal study to identify testicular genes whose expression is altered in YHR(+) mice during postnatal development. The mRNA expression levels for the steroidogenic enzymes, P450 17alpha-hydroxylase, 17beta-hydroxysteroid dehydrogenase3 and 5alpha-reductase1 were down-regulated in 3- and 5-week-old YHR(+) testis. This result coupled with an immunohistochemical analysis of Leydig cell specific proteins and quantification of Leydig cell numbers identified a decrease in adult Leydig cells in YHR(+) mice. Surprisingly, no change was detected for cytochrome P450 side-chain cleavage or steroidogenic acute regulatory protein RNA levels between WT and YHR(+) mice. In contrast, mRNA levels for insulin-like growth factor binding protein 3 were up-regulated in 3- and 5-week-old YHR(+) mice. The mRNA levels for several germ cell-specific proteins were up-regulated at 5 weeks of age in both WT and YHR(+) mice. We conclude that premature high levels of testosterone alter the expression of a select number of testicular genes and impair the differentiation of adult Leydig cells in mice.