The growth hormone receptor (GHR) is expressed in most cells throughout the body, and is a critical regulator of postnatal growth and metabolism in mammals. Many of its actions are mediated via its stimulation of insulin like growth factor-1 (IGF-1) expression. To date, much of the role of growth hormone signalling has been elucidated using in vitro cell-based assay systems, whilst in vivo knock-out models have emphasised the importance of GHR function for such endpoints as longitudinal growth, metabolism and reproductive fertility. In particular, several in vitro studies have investigated the regions of the cytoplasmic domain of the growth hormone receptor that are involved in recruiting the range of intracellular signalling proteins which are known to be stimulated by receptor activation upon ligand binding. By creating similar targeted mutations to the intracellular portion of the murine GHR, this thesis describes the creation and phenotype analysis of transgenic mouse models designed to elucidate the specific regions of the growth hormone receptor cytoplasmic domain that are required for its observed in vivo actions.
A targeting strategy was developed that utilised a genomic DNA clone encompassing exons 9 and 10 which code for the cytoplasmic domain of the growth hormone receptor. Interestingly, sequence analysis of the this clone revealed several coding anomalies when compared to published reports, suggesting more conservative amino acids when compared to other GHR species. This clone was subjected to mutagenesis to create five separate targeting constructs (Mutants 1 5), which were each designed to abrogate specific signalling pathways stimulated by the growth hormone receptor. These constructs were electroporated into 129/SVJ agouti ES cells to yield successfully recombined clones for all five targeting constructs.
Heterozygote targeted ES cells were injected into C57 black blastocysts to generate 13 chimeric mice throughout the duration of this project. Three of the male chimaeras reached germline in two of the mutant types created (Mutant 1 and Mutant 2). Heterozygous mice carrying germline transmission of both Mutant 1 (569 stop, Y539/545- F) and the more severe truncation Mutant 2 (391 stop) were mated to give rise to mendelian litter ratios of wild type, heterozygote and knock-in progeny.
Comparisons of growth rates for these litters showed progressive impairment of longitudinal growth corresponding to the severity of the mutation. A partial reduction in growth was also observed for both types of heterozygotes suggesting that the truncated receptors form inactive signalling units with full-length receptors when dimerised by growth hormone. These observations were in agreement with the relative organ weights and bone lengths recorded. Both lines displayed reduced serum IGF-1 levels, being more prominent for Mutant 2, which was comparible to GHR knockout mouse levels. Interestingly, reduced serum IGFBP-3 in mutant 1 may serve to impair the formation of large molecular weight IGFBP-3/ALS/IGF-q protein complexes in the serum, causing increased degradation of serum IGF-1 protein as evidenced by comparison with IGF-1 transcript levels.
Ability of mutants to generate STAT5 in response to growth hormone stimulation of prepubescent mice revealed that Mutant 1 was able to stimulate a submaximal Stat5 phosphorylation response, whilst Mutant 2 was not. It is likely that tyrosine 498 is able to stimulate a submaximal StatS response in vivo, whilst the receptor proximal tyrosines 341, 346,401 and 447 are insufficient to stimulate StatS. As growth hormone receptor stimulates sexually dimorphic liver gene expression through the cyclic activation of Stat5B in response to differential pulse patterns of growth hormone release from the pituitary indices of sexual dimorphism were examined. Levels of the sexually dimorphic major urinary protein (MUP) in the urine of male and female mice. Mutant 1 displayed a reduction in MUP levels in both males and females, whilst no MUP was detectable in urine taken from Mutant 2 as is observed for GHR knockout mice. Similarly, male specific cytochrome P4S0 liver transcript levels displayed female characteristics for mutant 2 only.
In conclusion this thesis presents the first in vivo models of mice expressing targeted mutations to the cytoplasmic domain of the growth hormone receptor. This work supports a critical role of the distal cytoplasmic domain of the growth hormone receptor for the stimulation of postnatal growth and sexually dimoiphic function whilst also suggesting a role for Stat5 in the regulation of IGF-1 and IGFBP-3 transcription.