Kinase-inactive G-protein-coupled receptor kinases are able to attenuate follicle-stimulating hormone-induced signaling.

Homologous desensitization of G-protein-coupled receptors (GPCR) is thought to occur in several steps: binding of G-protein-coupled receptor kinases (GRKs) to receptors, receptor phosphorylation, kinase dissociation, and finally binding of beta-arrestin to phosphorylated receptors and functional uncoupling of the associated Galpha protein. It has recently been reported that GRKs can inhibit Galphaq-mediated signaling in the absence of phosphorylation of some GPCRs. Whether or not comparable phosphorylation-independent effects are also possible with Galphas-coupled receptors remains unclear. In the present study, using the tightly Galphas-coupled FSR receptor (FSH-R) as a model, we observed inhibition of the cAMP-dependent signaling pathway using kinase-inactive mutants of GRK2, 5, and 6. These negative effects occur upstream of adenylyl cyclase activation and are likely independent of GRK interaction with G protein alpha or beta/gamma subunits. Moreover, we demonstrated that, when overexpressed in Cos 7 cells, mutated GRK2 associates with the FSH activated FSH-R. We hypothesize that phosphorylation-independent dampening of the FSH-R-associated signaling could be attributable to physical association between GRKs and the receptor, subsequently inhibiting G protein activation.

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.

Beta2 adrenergic receptors mediate cAMP, tissue-type plasminogen activator and transferrin production in rat Sertoli cells.

FSH is the main regulator of Sertoli cell function. Nevertheless, several other effectors such as catecholamines can also stimulate these cells through the adenylyl cyclase transduction pathway. However, the expression of beta adrenergic receptors in Sertoli cells is a subject of controversy. The aim of the present study was to determine if there are physiologically functional beta adrenergic receptors in Sertoli cells and to which subtype(s) they belong. In freshly isolated Sertoli cells, isoproterenol, a non selective beta-adrenergic agonist, was found to stimulate cAMP production and tissue-type plasminogen activator secretion. Specific transcripts for the beta1 and beta2, but not beta3, subtypes were detected by RT-PCR analysis. Beta2 transcripts were the form expressed predominantly in Sertoli cells. Binding experiments carried out on freshly isolated and on cytospined Sertoli cells indicated that in both conditions, [125I]iodocyanopindolol binding was inhibited by a non-selective and a 2 selective antagonist, whereas a beta1 selective antagonist had no effect. Scatchard analysis of beta2 specific inhibition revealed a dissociation constant of 0.3 nM and a receptor density of 14000 sites per cell. In freshly isolated Sertoli cells, we observed that cAMP and tissue-type plasminogen activator were stimulated by isoproterenol and a beta2 selective agonist, but not by beta1 or beta3 selective agonists. Accordingly, the isoproterenol-stimulated tissue-type plasminogen activator responses were abolished by the beta2 selective antagonist only. In cultured Sertoli cells, the trend was the same: tissue-type plasminogen activator and transferrin secretions were increased by isoproterenol and beta2 but not by beta1 or beta3 selective agonists. We conclude that freshly isolated Sertoli cells express beta2 adrenergic receptors which are functionally coupled to adenylyl cyclase and that these characteristics are preserved in cell culture. For the tested parameters, catecholamines and FSH effects were similar, but response magnitudes were systematically lower with beta agonists than with FSH. As norepinephrine is normally present in physiologically-relevant amounts in the interstitial fluid, it can be suspected to play a role in the regulation of Sertoli cell function.

Rat G protein-coupled receptor kinase GRK4: identification, functional expression, and differential tissue distribution of two splice variants.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G protein-coupled receptors, leading to the homologous mode of desensitization. We report here on the cloning of complementary DNAs that encode two rat GRK4 variants. Rat GRK4A (575 amino acids) displays 76% identity with the long human GRK4 splice variant. Rat GRK4B (545 amino acids) delineates a new variant that is identical to GRK4A except for a 31-amino acid deletion in the N-terminal domain, corresponding to exon VI in the human GRK4 gene. GRKs4A and B are likely produced by alternative splicing from a single gene, the partial characterization of which revealed a structural organization similar to that of the human GRK4 gene. GRK4A messenger RNA (mRNA) is abundant only in testis. A combination of in situ hybridization and quantitative RT-PCR studies demonstrated that GRK4A mRNA level increases during testicular development and predominates in leptotene to late pachytene primary spermatocytes and round spermatids. GRK4B mRNA is poorly expressed in testis and most rat tissues but is heterogeneously distributed in the kidney, with 20-fold enrichment in the outer medulla. GRKs4A and B are both functional protein kinases, as demonstrated in a rhodopsin phosphorylation assay. The differential tissue distribution of GRKA4 and GRK4B suggests that individual GRK4 variants may serve distinct physiological functions.

Expression of horse and donkey LH in COS-7 cells: evidence for low FSH activity in donkey LH compared with horse LH.

Horse (Equus caballus) luteinizing hormone (eLH) and chorionic gonadotrophin (eCG), which have the same amino acid sequence, are unusual in that, although they express only LH activity in equids, they express dual LH and FSH activities in all other species tested. Donkey (Equus asinus) LH (dkLH) and CG (dkCG), which also share an identical peptide backbone, have been less well characterized and conflicting results concerning their FSH activity in heterologous species have appeared in the literature. In order to assess and compare the intrinsic LH and FSH activities of the horse and donkey LHs in heterologous species, recombinant eLH (r.eLH/CG) and recombinant dkLH (r.dkLH/CG) were expressed, for the first time, in COS-7 cells. Their LH activities were assessed in a rat Leydig cell bioassay, and their FSH activities were estimated in a bioassay using Y1 cells stably expressing the human FSH receptor. Human CG (hCG) was expressed (r.hCG) and analysed in the same system. The results showed that, whereas r.dkLH/CG was about twice as active as r.eLH/CG in the LH bioassay, it was five times less active than r.eLH/CG in the FSH bioassay; r.hCG was about three times less active than r.eLH/CG in the LH bioassay but was completely inactive in the FSH bioassay. These results confirm that dkLH/CG possesses significant FSH activity in heterologous species that is not attributable to contamination with FSH.

Evidence that the alpha-subunit influences the specificity of receptor binding of the equine gonadotrophins.

Horse LH/chorionic gonadotrophin (eLH/CG) exhibits, in addition to its normal LH activity, a high FSH activity in all other species tested. Donkey LH/CG (dkLH/CG) also exhibits FSH activity in other species, but about ten times less than the horse hormone. In order to understand the molecular basis of these dual gonadotrophic activities of eLH/CG and dkLH/CG better, we expressed, in COS-7 cells, hybrids between horse and donkey subunits, between horse or donkey alpha-subunit and human CG beta (hCG beta), and also between the porcine alpha-subunit and horse or donkey LH/CG beta. The resultant recombinant hybrid hormones were measured using specific FSH and LH in vitro bioassays which give an accurate measure of receptor binding specificity and activation. Results showed that it is the beta-subunit that determines the level of FSH activity, in agreement with the belief that it is the beta-subunit which determines the specificity of action of the gonadotrophins. However, donkey LH/CG beta combined with a porcine alpha-subunit exhibited no FSH activity although it showed full LH activity. Moreover, the hybrid between horse or donkey alpha-subunit and hCG beta also exhibited only LH activity. Thus, the low FSH activity of dkLH/CG requires an equine (donkey or horse) alpha-subunit combined with dkLH/CG beta. These results provide the first evidence that an alpha-subunit can influence the specificity of action of a gonadotrophic hormone.