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Effect of deglycosylation on the structure and hormone-binding activity of the lutropin receptor.

ABSTRACT: Affinity-purified rat ovarian lutropin (LH) receptor is a single 90 kDa polypeptide which binds to immobilized lectins, indicating that the receptor is a glycoprotein [Keinänen, Kellokumpu, Metsikkö & Rajaniemi (1987) J. Biol. Chem. 262, 7920-7926]. In the present study the glycoprotein nature of the rat ovarian LH receptor was investigated in order to determine the contribution of the glycan moiety to receptor's size and hormone-binding properties. Treatment of the 125I-labelled purified LH receptor with neuraminidase and peptide N-glycosidase F resulted in a decrease in size of LH receptor from 90 kDa to 79 kDa and 62 kDa respectively, as assessed by SDS/polyacrylamide-gel electrophoresis. Endo-alpha-N-acetylgalactosaminidase treatment did not affect the electrophoretic mobility of the intact or neuraminidase-treated LH receptor. Subjecting the membrane-bound LH receptor to similar enzymic treatments followed by ligand blotting showed that the 79 kDa and 62 kDa forms are capable of specific hormone binding. Furthermore, intact and peptide N-glycosidase F-treated membranes bound 125I-labelled human choriogonadotropin with similar affinities. These data suggest that molecular mass of the polypeptide backbone of the LH receptor is 62 kDa. The receptor contains N-glycosidically linked oligosaccharide chains with terminal sialic acid residues, with little or no O-linked oligosaccharide. N-Linked carbohydrate is not required for specific high-affinity hormone binding.

SUBMITTER: Keinanen KP

PROVIDER: S-EPMC1135475 | biostudies-other | 1988 Dec

REPOSITORIES: biostudies-other

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Project description:Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning ?-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on "conformationally constrained" GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function.

Project description:The oligomeric glycoprotein hormone, ovine lutropin was treated with anhydrous HF at 0 degrees C for 30, 60 and 180 min and at 23 degrees C for 60 and 180 min. The products, designated deglycosylated lutropin 1 (DGLH-1) to deglycosylated lutropin 5 (DGLH-5) respectively, were characterized by gel filtration, concanavalin A-Sepharose binding, disc electrophoresis, amino acid analysis, carbohydrate composition and spectral properties. The preparations were also evaluated for receptor binding activity and immunological activity and bioassayed in vitro in collagenase-dispersed rat interstitial cells. In DGLH-1, fucose and galactosamine were removed completely, and there was a 94% decrease in hexoses and 39% decrease in N-acetylglucosamine. Reaction with HF at 0 degrees C for 1 or 3h led to removal of all hexoses and additional loss of hexosamines. Reactions at 23 degrees C for either 1 or 3h were not of additional value in deglycosylation and none of the reaction conditions yielded the apohormone. All the five deglycosylated hormone preparations were not retained on immobilized-concanavalin A columns and on Sephadex G-100 they were eluted with an increased V(e)/V(0) ratio consistent with the loss of carbohydrate residues. Loss of all but the last of the N-acetylglucosamine residues decreased the abnormality of lutropin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but did not eliminate it. Receptor binding activities of DGLH-1 and DGLH-2 were not different from that of the native hormone, but that of DGLH-3 was slightly decreased and the products obtained at 23 degrees C (DGLH-4 and DGLH-5) had lower activity. Immunoreactivities followed a similar pattern. None of the derivatives had activity in the bioassay in vitro. All of the five derivatives inhibited the action of the native hormone in the bioassay in vitro. Their hormonal antagonistic activity was consistent with the receptor binding activity, with DGLH-5 being the least potent in this respect. The DGLH-4 and DGLH-5 preparations had undergone conformational changes as revealed by 8-anilinonaphthalene-1-sulphonate fluorescence, but this did not result in loss of quaternary structure.

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Effect of deglycosylation on the structure and hormone-binding activity of the lutropin receptor.

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