Disruption of potential sites for N-linked glycosylation does not impair hormone binding to the lutropin/choriogonadotropin receptor if Asn-173 is left intact.

Abstract The rat lutropin/choriogonadotropin receptor (rLHR) is a G protein-coupled receptor, the large extracellular domain of which binds human choriogonadotropin (hCG) with high affinity. Within the extracellular domain are six potential sites for N-linked glycosylation. Although several studies have attempted to determine if N-linked carbohydrates are necessary for hormone binding, the results have been in apparent disagreement. In this study we have used site-directed mutagenesis to singly and collectively alter the consensus sequences for N-linked glycosylation in the rLHR. In particular, we examined the binding activity in both intact cells as well as detergent-solubilized extracts so that the effects on trafficking to the plasma membrane could be determined. In addition, we independently examined the effects of substituting a particular Asn versus Thr or Ser residue within a given glycosylation consensus sequence. Our data suggest that substitution of Asn-173 with Gln results in both a decreased ability of the receptor to be expressed on the plasma membrane as well as a vastly decreased binding affinity of the receptor for hCG. However, if the consensus sequence for N-linked glycosylation at Asn-173 is altered by substitution of Thr-175 with Ala (instead of Asn-173 to Gln), the resulting receptor binds hCG with high affinity although it is still impaired in its ability to be expressed at the plasma membrane. Furthermore, if all consensus sequences for N-linked glycosylation are mutated collectively while maintaining Asn-173 (by substituting Thr-175 with Ala instead of Asn-173 to Gln), the resulting deglycosylated receptor, although not expressed on the plasma membrane, binds hCG with high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)