IN-VITRO MUTAGENESIS OF GROWTH-HORMONE RECEPTOR ASN-LINKED GLYCOSYLATION SITES

Site-directed mutagenesis was used to replace asparagine (Asn) residue s with glutamine (Gln) at the five potential N-linked glycosylation si tes located at positions 28, 97, 138, 143, and 182 in the extracellula r domain of the porcine growth hormone receptor (pGHR). These mutated pGHR cDNAs were stably expressed in mouse L cells. Single substitution of the Asn residues did not alter growth hormone binding when compare d to cells which express native-pGHR (K-D = 1 nM). However, substituti on of the five potential Asn-linked sites together (pGHR Delta 5) resu lted in a 20-fold reduced GH binding affinity (K-D = 20 nM). Residues Asn(97), Asn(138), and Asn(182) were apparently glycosylated and upon cross-linking with I-125-labeled pGH migrated as a molecular complex o f approximately 130 kDa. Native pGHR and pGHR analogs with substitutio ns of N28Q and N143Q when cross-linked to I-125-labeled pGH, migrated with a M(r) of 138 kDa. The fully deglycosylated cross-linked receptor , pGHRL Delta 5, migrated as a complex of 108 kDa. Therefore, each car bohydrate moiety contributed approximately 10 kDa to the total molecul ar mass of the pGHR, in sum contributing 30 kDa to the total M(r) of t he glycosylated pGHR. pGHR Delta 5 was able to internalize nearly all the bound I-125-labeled pGH within 10 min, whereas native pGHR and ind ividual Asn substituted pGHR analogs internalized 25% of bound I-125-l abeled GH at 10 min. Also, mutagenesis of the pGHR five potential Asn- linked glycosylation sites, either singly or together, did not alter t he ability of GH to induce tyrosine phosphorylation of a 95-kDa protei n. Together, the results indicate that three of the five pGHR Asn resi dues are apparently glycosylated and are necessary for maintenance of a high affinity GH binding site and for GH internalization. However, g lycosylation of the pGHR is not critical for eliciting tyrosine phosph orylated proteins following the GH/GHR interaction.