Phosphorylation and glycosylation of the luteinizing hormone receptor.

Abstract

Purified testicular and ovarian luteinizing hormone/human chorionic gonadotropin (hCG) receptors are phosphorylated at serine and threonine residues by the catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). Occupancy of the receptors by hCG significantly increased the rate but not the extent of phosphorylation. However, prolonged preincubation of receptors with hCG reduced the subsequent rate of receptor phosphorylation. Identical phosphopeptide maps were obtained for the phosphorylated ovarian and testicular receptors. The phosphorylated receptor, like the native receptor, bound to wheat germ lectin and hCG-Sepharose and migrated as a single band of Mr 90,000 (testis) and Mr 85,000 (ovary) on NaDodSO4/PAGE.Neuraminidase treatment of receptors caused reductions of molecular weight to 82,000 (testis) and 77,000 (ovary), and further treatment with O-Glycanase had mnimal effect on molecular size. However, deglycosylation with N-Glycosidase and endoglycosidase F produced a single labeled polypeptide of Mr 59,000 for both gonadal receptors. Treatment of native receptors with neuraminidase caused no apparent change in binding of gonadotropin to blotted receptors, whereas deglycosylated receptors showed a major reduction in hormone binding. These results indicate that luteinizing hormone/hCG receptors are sialoglycoproteins with predominantly N-linked glycosyl residues that account for the size difference between testicular and ovarian receptors and that may participate in the interaction with gonadotropin. Receptor occupancy by agonist leads to a conformational change that facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to hCG.The luteinizing hormone (lutropin, LH) receptor from rat testis and ovary has been purified by sequential steps of affinity-column chromatography and demonstrated to be a single protein species that binds LH and human chorionic gonadotropin (hCG) (1, 2). The purified LH/hCG receptor has been shown to be a substrate for phosphorylation by cAMP-dependent protein kinase (protein kinase A) (2). The phosphorylated receptor retains its binding affinity for 125I_ labeled hCG and can be employed for structural studies when only minute quantities of receptor protein are available for analysis.Detailed structural characterization of the receptor will be essential to understanding the mechanism of action of gonadotropins. Also, it is conceivable that glycosylation is responsible for the size differences observed for LH/hCG receptors from testis and ovary (1,2 Phosphorylation of Receptor. The solubilized ovarian and testicular receptors were purified to electrophoretic homogeneity and phosphorylated as described (1, 2). Briefly, 1 pmol of purified receptor was added, either directly or after preincubation with hCG, to a reaction mixture containing 1 unit of catalytic subunit of protein kinase A, 10 mM Tris HCI, 10 mM MgCl2, 0.3 mM dithiothreitol, and 10 ,uM [y-32P]ATP (final pH, 7.4). The phosphoryla...

Functional Glycosylation Sites of the Rat Luteinizing Hormone Receptor Required for Ligand Binding

“…In contrast, the carbohydrate chains of the high affinity rat ovarian LHR are not high mannose but are rather of the complex type (2). This suggests that either carbohydrate residues common to both the high mannose and complex chains are central to the formation of the hormone binding domain, or that carbohydrate function occurs in the mammalian cell prior to processing of the high mannose chains to the complex form, as has been reported with the mannose 6-phosphate receptor (17).…”

“…A functional role for N-linked carbohydrates in high affinity hormone binding has not yet been established, and is a subject of some controversy. Conflicting reports on the effects of deglycosylation (2,4) and site-directed mutagenesis (5,6) of the mature holoreceptor on hormone binding activity may be a function of the original receptor state. N-Linked carbohydrate chains of the rat ovarian LH receptor have previously been shown to be essential for high affinity hormone binding by a number of different methods, including site-directed mutagenesis of the Asn of the putative glycosylation sites (5), tunicamycin treatment (7), and enzymatic deglycosylation of solubilized receptors (2).…”

“…Conflicting reports on the effects of deglycosylation (2,4) and site-directed mutagenesis (5,6) of the mature holoreceptor on hormone binding activity may be a function of the original receptor state. N-Linked carbohydrate chains of the rat ovarian LH receptor have previously been shown to be essential for high affinity hormone binding by a number of different methods, including site-directed mutagenesis of the Asn of the putative glycosylation sites (5), tunicamycin treatment (7), and enzymatic deglycosylation of solubilized receptors (2). These procedures have not resolved the question of whether the reported carbohydrate requirement of the receptor is based on a direct interaction with the hormone or is related to the conformation of the ligand binding site.…”

“…It is noteworthy that glycoproteins in insect cells have been shown to carry N-linked carbohydrates of the high mannose type that are trimmed to shorter core structures (Man 3 GlcNAc 2 ) (9). In contrast, the mammalian cell, which has been shown to express high-affinity LHR (8), carries a full complement of trimming and complex glycosidases in its post-translational machinery (2). The present experiments demonstrate a post-translational glycosylation requirement that is satisfied by high mannose carbohydrate chains.…”

“…1) and N-linked carbohydrate (CHO) chains of the complex type (1)(2)(3). A functional role for N-linked carbohydrates in high affinity hormone binding has not yet been established, and is a subject of some controversy.…”