Sensitivity of hybrid ovine/rat GH receptors to oGH and rat GH in transfected FDC-P1 mouse myeloid cells in vitro.
We have described previously the properties of two mutant ovine growth hormone receptor extracellular domain (oGHR-ECD) proteins which were created by substituting sequences from the rat GHR at two different locations within the framework of the oGHR-ECD. The first mutation occurred at a region close to the N-terminus of oGHR-ECD between residues Thr 28 and Leu34 and created the protein T28E/N29S/N33K/L34P-oGHR-ECD, where the ovine specific residues T, N, N and L are replaced by their equivalent residues E, S, K and P from the rat protein. This site lies N-terminal to the first element of beta-strand structure in the GHR-ECD and we designated this protein as Site-A mutant. The second mutation was made between residues Serl21 andAsp 124 of oGHR-ECD to produce the protein S121T/E123D/D124E-oGHR-ECD where ovine specific residues are again replaced with the equivalent residues from the rat GHR-ECD. This region lies in a loop structure which joins the two beta-barrel domains of the GHR-ECD. This protein is designated as Site-B mutant. A subsequent analysis confirmed the N-terminal region between residues 28-34 of oGHR-ECD as an important epitope defined by antiserum raised to oGHR-ECD. Also of interest was the finding that mutation at both Sites A and B in oGHR-ECD compromised the affinity of the protein for bovine GH (50-fold for Site-A and 4-fold for Site-B). A comparison of the affinity of wild type oGHR for the highly homologous bovine GH with its affinity for rat GH indicated a 10-fold higher affinity for the ruminant GH than for rat GH. Mutation at Site-A of oGHR-ECD reduced the affinity for rat GH a further 3-fold. However, mutation at Site-B of oGHR-ECD increased the affinity for rat GH 2-3 fold. This indicated that the substitution of rat GHR residues for ovine GHR residues in this part of the protein had a beneficial effect in relation to affinity for rat GH and that this region of the GHR-ECD may contain important specificity determinants. In order to test whether these observed differences in affinity for bovine and rat GH affinities have any biological relevance, we have produced the same ovine --> rat mutations in the context of the full length ovine GHR. Transfection of the cDNAs encoding the wt or mutant GHRs into the mouse myeloid pre-B cell line FDC-P1 to create stably transfected clonal lines, has allowed an examination of the relative activities of ovine and rat GH, using a robust and high throughput bio-assay based on the reduction of a cell permeable tetrazolium salt. In the current manuscript, we report that the decrease in binding affinity previously reported for Site-A and Site-B mutant oGHR extracellular domain proteins is not reflected in compromised biological activities when the same mutations are expressed in the context of the full length oGHR protein. We discuss these findings in the context of the relationships between affinity and activity at the GHR.
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D013006:Growth Hormone / Q000494:pharmacology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D022423:Myeloid Cells / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D013045:Species Specificity; D014162:Transfection
D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone