Study of goldfish (Carassius auratus) growth hormone structure-function relationship by domain swapping.
Using goldfish as a model, the structure-function relationship of goldfish growth hormone was studied using the strategy of homologous domain swapping. Chimeric mutants were constructed by exchanging homologous regions between goldfish growth hormone (gfGH II) and goldfish prolactin (gfPRL) with their cloned complementary DNAs. Six mutants, with their domain-swapped, were generated to have different combinations of three target regions, including the helix a, helix d and the large section in between these helices (possess the helices b, c and other random coiled regions). After expression in E. coli and refolding, these mutants were characterized by using competitive receptor binding assay (RRA) and growth hormone responding promoter activation assay. The different activity profiles of mutants in Spi 2.1 gene promoter assays from that in RRA shows that, for gfGH, receptor binding dose not confer receptor signal activations. When either helices a or d of gfGH was maintained with other helices replaced by their gfPRL counterparts, both receptor binding and hence gene activation activities are reduced. In mutants with helices b and c in gfGH maintained, containing the gfGH middle section, and helices a and d swapped with gfPRL, the had reduced RRA activities but the promoter activation activities retained. In conclusion, as in the case of human GH, the gfGH molecule possesses two functional sites: one of them is composed of discontinuous epitopes located on the target regions of this study and is for receptor binding; another site is located on the middle section of the molecule that helices a and d are not involved, and it is for activation of GH receptor and intracellular signals.
Chan
Y H
YH
Department of Biochemistry, Chinese University of Hong Kong, Sha Tin, New Territories, Hong Kong SAR, China.
Cheng
C H K
CH
Chan
K M
KM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006054:Goldfish / Q000378:metabolism*; D013006:Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
C091103:LOC299282 protein, rat; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D013006:Growth Hormone