N-Linked Oligosaccharides Are not Required for Hormone Binding of the Lutropin Receptor in a Leydig Tumor Cell Line and Rat Granulosa Cells

Abstract

We have examined roles of carbohydrates of the lutropin receptor in a murine Leydig tumor cell line (MLTC) and primary cultures of rat granulosa cells. We approached this issue by deglycosylating mature receptors with glycosidases and by preventing glycosylation of nascent receptors with tunicamycin B2, an inhibitor of protein glycosylation but not protein synthesis. Deglycosylation of mature receptors with neuraminidase, N-glycanase or both did not affect ligand binding capacity. Regardless of glycosidase treatment, the number of hormone binding sites was similar. The Kas for native receptors, asialoreceptors and aglycoreceptors, are also comparable, being 2.0 x 10(9) M-1, 1.9 x 10(9) M-1 and 1.7 x 10(9) M-1 respectively. In contrast, cells treated with tunicamycin B2 failed to bind the hormone. These results demonstrate that N-oligosaccharides of mature lutropin receptors are not required for ligand binding. In addition, our data suggest, for the first time, that N-glycosylation of the receptor may be necessary for expressing functional receptors on the cell surface and that there exist striking similarities in roles of oligosaccharides of lutropin and its receptor.

Glycosylation of the Gastrin-Releasing Peptide Receptor and Its Effect on Expression, G Protein Coupling, and Receptor Modulatory Processes

“…In a study using enzymatic deglycosylation, GRP-R carbohydrate was proposed to be involved in regulating this receptor's ability to couple to G proteins (Kusui et al, 1994). This finding is similar to that obtained from studies of the VIP receptor but is in contrast to that obtained for the LH (Ji et al, 1990;Petaja-Repo et al, 1993), somatostatin (Rens-Domiano and Reisine, 1991), and M1, M2, and M4 muscarinic cholinergic receptors (Ohara et al, 1990). Few G protein-coupled, seven transmembrane-spanning receptors have been studied at the molecular level to determine whether glycosylation is involved in G protein activation.…”

“…Although the receptors for neurotransmitters, such as adrenergic and cholinergic agents (Ohara et al, 1990;Rands et al, 1990;Giovannelli et al, 1991), and the receptors for classical hormones including LH and TSH (Ji et al, 1990;Russo et al, 1991;Zhang et al, 1991;Liu et al, 1993;Petaja-Repo et al, 1993), have been extensively studied using both traditional pharmacological and molecular biological techniques, relatively little is known about the nature or importance of carbohydrate for the receptors for gastrointestinal hormones including the GRP-R. From cross-linking studies and timed PNGase F digestions, it was proposed that the GRP-R possesses four separate extracellular carbohydrate residues (Kusui et al, 1994) and that the glycosylation of this receptor was of critical importance for maintaining highaffinity binding and mediating G protein coupling (Kusui et al, 1994). However, no direct data were provided as to whether each glycosylation consensus sequence was in fact glycosylated, the exact amount of carbohydrate attached to each consensus sequence, or whether these alterations in binding and G protein coupling consequent to receptor deglycosylation could be attributed to glycosylation of any particular consensus sequence.…”

Glycosylation of G-protein-coupled receptors for hormones central to normal reproductive functioning: its occurrence and role

“…The precise role of the oligosaccharide moieties of the HCG/LHR is controversial, as data published by various laboratories appear to be mutually exclusive. For example, hormone binding was reported to be unaffected by enzymatic de-glycosylation of the mature receptor (Keinänen, 1988;Ji et al, 1990;Patäjä-Repo et al, 1993;Tapanainen et al, 1993) but to be decreased by disruption of three of the glycosylation sites (Asn 77 , Asn 152 and Asn 173 ) using site-directed mutagenesis (Zhang et al, 1991). This decreased ligand binding was not due to compromised expression of the receptor at the cell surface.…”

Molecular structures of glycoprotein hormones and functions of their carbohydrate components

“…Hormone obtained from pooled urine could well contain carbohydrate derived from malignant tissue [248] and while glycosylation patterns may not be identical with those of human origin, recombinant species if available in sufficient quantity may yield more consistent information [249] and provide a structurally well-defined molecule whose carbohydrate and protein domains could be more accurately related to biological activities such as receptor-binding [250] and changes associated with the hormonal response [251,252].…”