THE 6 N-LINKED CARBOHYDRATES OF THE LUTROPIN CHORIOGONADOTROPIN RECEPTOR ARE NOT ABSOLUTELY REQUIRED FOR CORRECT FOLDING, CELL-SURFACE EXPRESSION, HORMONE-BINDING, OR SIGNAL-TRANSDUCTION/

Using two separate methods, we have determined that all six potential sites for N-linked glycosylation on the rat lutropin/choriogonadotropi n receptor (rLHR) contain carbohydrates. The functional roles of the c arbohydrates were analyzed initially through the use of two nonglycosy lated receptor mutants rLHR(N(77,152,173,269,277,291)Q) and rLHR(N(77, 152,269,277,291)Q;T(175)A). Although Western blot analyses demonstrate d both mutant receptors to be stably expressed, little or no hCG bindi ng activity could be detected in detergent solubilized extracts of 293 cells expressing either nonglycosylated LHR mutant. Although this los s of hCG binding was concluded to be due to misfolding, it was unknown whether this misfolding was due to the absence of carbohydrates or to the multiple amino acid substitutions that had been introduced into t he polypeptide. To differentiate between these possibilities, hCG bind ing assays were performed with nonglycosylated receptors obtained afte r tunicamycin treatment of cells expressing the wildtype rLHR. Even th ough these wild-type receptors were confirmed to be devoid of all N-li nked carbohydrates by Western blots, they were found to bind hCG with a normal high affinity. In addition, tunicamycin-derived, nonglycosyla ted LHRs were present at the cell surface and exhibited a phenotype co nsistent with mature receptors due to their capability to mediate hCG- stimulated cAMP production as well as bind oLH with high affinity. The se results indicate that the loss of high affinity hormone binding by rLHR(N(77,152,173,269,277,291)Q) and rLHR(N(77,152,269,277,291)Q;T(175 )A) is simply due to the collective amino acid substitutions rather th an to the absence of carbohydrates. Therefore, N-linked carbohydrates are not absolutely required for the proper folding of the rLHR into a mature receptor capable of binding hormone and signaling. These result s are in marked contrast to the follitropin receptor (FSHR), a very si milar receptor which has been shown to strictly require N-linked carbo hydrates for folding of the nascent protein.