Mario Ascoli

Signaling and Phosphorylation-impaired Mutants of the Rat Follitropin Receptor Reveal an Activation- and Phosphorylation-independent but Arrestin-dependent Pathway for Internalization

We have previously shown that the rat follitropin receptor (rFSHR) expressed in transfected cells becomes phosphorylated upon stimulation of the cells with agonist or a phorbol ester. Peptide mapping and mutagenesis studies have also shown that the agonist- or phorbol ester-induced phosphorylation of the rFSHR maps to Ser/Thr residues present in the first and third intracellular loops. The experiments presented herein were initially designed to test for the presence of additional phosphorylation sites on the second intracellular loop of the rFSHR. Analysis of two new mutants in which the two threonines in the second intracellular loop (rFSHR-2L) or the two threonines in the second intracellular loop and the seven Ser/Thr residues in the third intracellular loop (rFSHR-2L + 3L) were mutated showed that one or more of the two threonines in the second intracellular loop are phosphorylated in response to phorbol ester, but not in response to agonist stimulation. Since rFSHR-2L and rFSHR-2L + 3L displayed a reduction in agonist-induced signaling, two additional mutants (rFSHR-D389N and rFSHR-Y530F) were constructed in an attempt to better understand the relationship between the agonist-induced activation, phosphorylation, and internalization of the rFSHR. These point mutations impaired agonist-stimulated signal transduction and abolished agonist-induced phosphorylation. Co-transfection studies revealed that the phosphorylation of these mutants can be rescued by overexpression of G protein-coupled receptor kinase 2, but this increased phosphorylation only rescues the internalization of rFSHR-D389N. The internalization of both mutants could be rescued by overexpression of arrestin-3, however. Taken together, these results argue that the agonist-induced activation and phosphorylation of the rFSHR are not essential for internalization. while the interaction of the rFSHR with a nonvisual arrestin is essential for internalization.

Truncation of the C-terminal Tail of the Follitropin Receptor Does Not Impair the Agonist- or Phorbol Ester-induced Receptor Phosphorylation and Uncoupling

We have recently shown that addition of follitropin (FSH) or a phorbol ester (phorbol 12-myristate 13-acetate (PMA)) to cells expressing the recombinant follitropin receptor (FSHR) results in both phosphorylation and uncoupling of the FSHR from adenylyl cyclase. In the light of findings reported with other G protein-coupled receptors we have proposed that phosphorylation of the FSHR mediates the uncoupling from adenylyl cyclase. The experiments described herein represent the first attempt to determine the location of the amino acid residues that become phosphorylated in FSHR and to test the hypothesis that phosphorylation is responsible for uncoupling of FSHR from adenylyl cyclase. As a first step in identifying which residues may be phosphorylated in response to hFSH and PMA, we constructed a mutant of the FSHR cDNA in which the C-terminal cytoplasmic tail was truncated at residue 635 (FSHR-t635), thus removing all but one of the potential phosphorylation sites present in the C-terminal tail. Cells expressing FSHR-t635 bind hFSH with the appropriate affinity and respond with increases in cAMP and inositol phosphate accumulation. The maximal cAMP and inositol phosphate responses of cells expressing FSHR-t635 are higher than those of cells expressing the wild type FSHR, but the concentration of hFSH required to elicit these responses is similar in both cell lines. Immunoprecipitation of FSHR-t635 shows that the truncated receptor is still effectively phosphorylated in response to hFSH or PMA. Phosphoamino acid analysis reveals that, like the wild-type FSHR, FSHR-t635 phosphorylation occurs on serine and threonine residues. Peptide mapping suggests that the phosphorylated residues in the FSHR and FSHR-t635 are located within the same areas of the intracellular regions of the receptors. In addition to stimulating phosphorylation of FSHR-t635, hFSH and PMA also effectively uncouple the truncated receptor from adenylyl cyclase. Taken together, these data show that hFSH and PMA can both phosphorylate and uncouple a FSH receptor species with a cytoplasmic tail truncated at residue 635.

Mutation of Individual Serine Residues in the C-terminal Tail of the Lutropin/Choriogonadotropin Receptor Reveal Distinct Structural Requirements for Agonist-induced Uncoupling and Agonist-induced Internalization

We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex. We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20–40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters. Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct.

Seven non-contiguous intracellular residues of the lutropin/choriogonadotropin receptor dictate the rate of agonist-induced internalization and its sensitivity to non-visual arrestins.

The amino acid sequences of the human (h) and rat (r) lutropin/choriogonadotropin receptors (LHR) are 87% identical, but the rate of agonist-induced internalization of the hLHR is ∼7 times faster than that of the rLHR. Chimeras of the hLHR and the rLHR showed that this rate is dictated by the serpentine domain and the cytoplasmic tail. Further mutational analysis identified seven residues, two adjacent residues in the second intracellular loop (Val/Gln in the rLHR and Ile/His in the hLHR), four non-contiguous residues in the third intracellular loop (Arg/Gln/Thr/Pro in the rLHR and Lys/Arg/Met/Thr in the hLHR), and one in the C-terminal tail (Leu in the rLHR and Phe in the hLHR), that are necessary and sufficient to impart the slow rate of internalization of the rLHR and the fast rate of internalization of the hLHR. The internalization of the rLHR and the hLHR display different sensitivities to the non-visual arrestins. Therefore, we also tested if the simultaneous exchange of these seven residues resulted in the exchange of this property. Since this was found to be the case, we propose that these seven residues identified here form a non-visual arrestin-binding site.

Intracellular uptake and catabolism of lutropin by testicular tissue in vivo

The mechanisms involved in the expression of biological activity of lutropin have been extensively studied [l] and, analogous to other peptide hormones, the initial step is believed to involve binding of the hormone to a specific receptor located in the plasma membrane of the target cells. The specificity and characteristics of hormone binding to testicular [ 1 ] and ovarian [2] receptors have been investigated and preparations of partially purified receptor are available [2-41. Little is known, however, about the fate of the hormone-receptor complex and its relationship, if any, to the termination of biological activity. Interestingly, it has recently been reported that the number of rat testicular gonadotropin receptors decreased with time following injection of either lutropin or choriogonadotropin [5]. Studies in this laboratory have been concerned with the kinetics and mechanisms of plasma clearance and biotransformations of lutropin [6-IO]. The results of in vivo studies on the kinetics of lutropin uptake and catabolism by target tissue are presented in the present communication. As expected, tritium labeled ovine lutropin binds specifically to the testes following intravenous injection into mature male rats. From sucrose gradient centrifugation, it appears that at least some of the hormones which are bound to Leydig cells are internalized and eventually degraded by lysosomes. This phenomenon suggests a possible mechanism for terminating hormone action by target cells and may be important in the regulation of lutropin receptors by the gonadotropin itself.

Structure of the lutropin/choriogonadotropin receptor

In summary, the LH/CG receptor is a single polypeptide which contains a large hydrophilic domain that is situated extracellularly, attached to a region that spans the plasma membrane seven times, the carboxy-terminal region being intracellular. This topology was predicted by the amino acid sequence and has been confirmed by our immunofluorescence studies. The extracellular domain, which is related to a family of leucine-rich glycoproteins, is presumably involved in binding the large glycoprotein hormones hCG and LH. The carboxy-terminal half of the receptor, which is related to the family of rhodopsinlike receptors, is (by analogy with these receptors) presumably involved in the coupling of the receptor to the G protein. Our transfection studies confirm that this single polypeptide is capable of binding hormone and activating adenylyl cyclase. Therefore, not only is the structure of the LH/CG receptor unique compared to other cell surface receptors characterized to date, but also its structure suggests that the mechanism of the translation of hormone binding to G protein coupling in this receptor is different from other G protein-coupled receptors whose ligands are much smaller and intercalcate among the transmembrane helices. We predict that, due to the homology among the glycoprotein hormones, the structures of the FSH and TSH receptors share extensive amino acid and structural homology with the LH/CG receptor. Last, our newly acquired knowledge about the structure of the LH/CG receptor, and the development of a cDNA and antibodies for this receptor, should enable more detailed studies on the function and regulation of the LH/CG receptor, not previously possible.

Ovulation Involves the Luteinizing Hormone-Dependent Activation of Gq/11 in Granulosa Cells

The LH receptor (LHR) activates several families of heterotrimeric G proteins, but only the activation of Gs and subsequent generation of cAMP are universally accepted as important mediators of LH actions. To examine the involvement of the Gq/11 family on the actions of LH, we crossed Cyp19Cre and Gαq(f/f);Gα11(-/-) mice to generate mice with a granulosa cell-specific deletion of Gαq in the context of a global deletion of Gα11. Granulosa cells from Gαq(f/f);Gα11(-/-);Cre(+) mice have barely detectable levels of Gαq/11, have a normal complement of LHR, and respond to LHR activation with a transient increase in cAMP accumulation, but they fail to respond with increased inositol phosphate accumulation, an index of the activation of Gαq/11. The LHR-provoked resumption of meiosis, cumulus expansion, and luteinization are normal. However, the Gαq(f/f);Gα11(-/-);Cre(+) mice display severe subfertility because many of the oocytes destined for ovulation become entrapped in preovulatory follicles or corpora lutea. Because follicular rupture is known to be dependent on the expression of the progesterone receptor (Pgr), we examined the LHR-induced expression of Pgr and 4 of its target genes (Adamts-1, Ctsl1, Edn2, and Prkg2). These actions of the LHR were impaired in the ovaries of the Gαq(f/f);Gα11(-/-);Cre(+) mice. We conclude that the defect in follicular rupture is secondary to the failure of the LHR to fully induce the expression of the Pgr. This is the first conclusive evidence for the physiological importance of the activation of Gq/11 by the LHR and for the involvement of Gαq/11 in ovulation.

Reactive Oxygen Species (ROS) Play a Critical Role in the cAMP-Induced Activation of Ras and the Phosphorylation of ERK1/2 in Leydig Cells

Activation of the LH receptor (LHR) in Leydig cells results in the phosphorylation of ERK1/2 by cAMP-dependent and cAMP-independent pathways. Here we examine the mechanisms by which cAMP stimulates ERK1/2 phosphorylation. We show that the stimulation of steroidogenesis is not necessary or sufficient to stimulate the phosphorylation of ERK1/2 but that other cAMP-dependent mitochondrial functions are involved. Using MA-10 cells as a model, we showed that cAMP analogs increase reactive oxygen species (ROS) formation and that an uncoupler of oxidative phosphorylation and a ROS scavenger prevent this increase. These two compounds also inhibit the increase in ERK1/2 phosphorylation provoked by cAMP analogs, thus suggesting that the cAMP-induced phosphorylation of ERK1/2 is mediated by mitochondrial ROS. In agreement with this hypothesis we also show that a reduction in glutathione levels, which alters the redox state of MA-10 cells, potentiates the effect of cAMP on ERK1/2 phosphorylation. Measurements of the dephosphorylation of ERK and the activation of Ras showed that the ROS scavenger prevents the cAMP-provoked activation of Ras and that cAMP, with or without a ROS scavenger, has little or no effect on the dephosphorylation of ERK. Lastly, we show that the uncoupler of oxidative phosphorylation and the ROS scavenger also prevent the ability of cAMP analogs to increase ERK1/2 phosphorylation in primary cultures of mouse Leydig cells. We conclude that, in Leydig cells, cAMP enhances the phosphorylation of ERK1/2 via a mitochondria-derived, ROS-dependent activation of Ras.

The Leydig Cell MEK/ERK Pathway Is Critical for Maintaining a Functional Population of Adult Leydig Cells and for Fertility

MAPK kinase (MEK)1 and MEK2 were deleted from Leydig cells by crossing Mek1(f/f);Mek2(-/-) and Cyp17iCre mice. Primary cultures of Leydig cell from mice of the appropriate genotype (Mek1(f/f);Mek2(-/-);iCre(+)) show decreased, but still detectable, MEK1 expression and decreased or absent ERK1/2 phosphorylation when stimulated with epidermal growth factor, Kit ligand, cAMP, or human choriogonadotropin (hCG). The body or testicular weights of Mek1(f/f);Mek2(-/-);iCre(+) mice are not significantly affected, but the testis have fewer Leydig cells. The Leydig cell hypoplasia is paralleled by decreased testicular expression of several Leydig cell markers, such as the lutropin receptor, steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, 17α-hydroxylase, and estrogen sulfotransferase. The expression of Sertoli or germ cell markers, as well as the shape, size, and cellular composition of the seminiferous tubules, are not affected. cAMP accumulation in response to hCG stimulation in primary cultures of Leydig cells from Mek1(f/f);Mek2(-/-);iCre(+) mice is normal, but basal testosterone and testosterone syntheses provoked by addition of hCG or a cAMP analog, or by addition of substrates such as 22-hydroxycholesterol or pregnenolone, are barely detectable. The Mek1(f/f);Mek2(-/-);iCre(+) males show decreased intratesticular testosterone and display several signs of hypoandrogenemia, such as elevated serum LH, decreased expression of two renal androgen-responsive genes, and decreased seminal vesicle weight. Also, in spite of normal sperm number and motility, the Mek1(f/f);Mek2(-/-);iCre(+) mice show reduced fertility. These studies show that deletion of MEK1/2 in Leydig cells results in Leydig cell hypoplasia, hypoandrogenemia, and reduced fertility.

Transfected cells express mostly the intracellular precursor of the lutropin/choriogonadotropin receptor but this precursor binds choriogonadotropin with high affinity.

Previous studies from several laboratories have shown that the cell surface rLHR is a 85-92 kDa protein synthesized from a 68-73 kDa intracellular precursor. While all investigators agree that the cell surface rLHR binds hCG with high affinity, it is not clear if the intracellular precursor can also bind hCG. In order to directly determine if the intracellular rLHR present in cells transfected with the wild-type rLHR binds hCG with high affinity, we devised a method that selectively degrades the cell surface rLHR while preserving the intracellular rLHR. The binding of hCG to intact cells was completely lost following mild proteolysis of the cells, but binding to detergent extracts prepared from proteolyzed cells was largely preserved. Measurements of the hCG binding affinity to intact cells or to detergent extracts prepared before and after proteolysis display very similar or identical binding affinities. Since binding to nonproteolyzed intact cells, detergent extracts prepared from nonproteolyzed cells, or detergent extracts prepared from proteolyzed cells occurs only to the 85-92 kDa rLHR, the 85-92 and 68-73 kDa rLHR, and the 68-73 kDa rLHR, respectively, we conclude that the cell surface rLHR and the intracellular rLHR bind hCG with the same affinity. Quantitation of the relative abundance of the cell surface and intracellular rLHR by immunological methods indicates that transfected cells express mostly the intracellular precursor. A comparison of the binding capacity of control and proteolyzed cells with that of their detergent extracts indicates that hCG binding assays greatly underestimate the relative abundance of the intracellular rLHR.