| Brain peptide CRF and CRFR1 are of important components of HPA axis, and have diverse responsivity to hypoxic challenge in the Tibetan native mammals of Ochotona curzoniae(OC), Microtus oeconomus(MO), and Myospalax baileyi(MB). We here report the clone and molecular evolution of those mammalian CRF,Ucn and CRFRla, CRF/CRFRla binding properties, and cellular cyclic AMP production and water permeability of CHO cells mediated by different CRFRla.We found that the coding region of CRFs from OC and MB are 41-aa peptide, the identity of the three mature proteins is 100%. The coding region of Ucn from MB and MO are 369 and 372bp, coding for a 122 and 123-aa proprepeptide, respectively. But both of these two proprepeptides code for a 40-aa mature peptide. The mature peptide of Ucn from MB is identical to that form mouse/rat, but that from MO have three aa difference with that from mouse/rat. CRFRla is 1269 bp coding for a 415-aa. Amino acid sequence showed that CRFRla in OC has identities of 96,95,95,95, and 95% with human, MO, MB, rat, and mouse. Molecular phylogenic clustering of CRFRla sequences in vertebrates including Tibetan's is consistent with the vertebrate morphological classification. Compared with lowland mammals, the CRFRla genes in Tibetan's have more mutated sites located within various domains. These mutated sites occur in the receptor-binding-domain and at extracellular and transmembrane region, as well as in potential PKC binding sites at the C-terminal in MO, the N-terminal and intracellular domain in MB, and the N-terminal and extracellular domain in OC. To further address the significance of these mutation, we constructed three wild-type CRFRla expression plasmids for those Tibetan animals and mice, rat and rabbit controls, then all were transfected into CHO cells to generate ocCRFR1-, moCRFR1-, mbCRFR1-, mCRFR1-, rCRFR1- and bCRFR1-CHO lines. We found that binding affinity of CRF/CRFRlas from the plateau mammals is lower than that of sea-level lived mouse. CRF and Ucn challenge concentration-dependent enhanced the cAMP production with similar pattern in media of these cells with oc-, mo-, mb-, and mouse CRFR1α, and the pattern of plateau mammals in EC50 values for CRF and for Ucn is in agreement with above binding data, when the cyclic AMP production in CHO cells transiently transfected with different CRFR1αs were determined. cAMP level stimulated by CRF was highest in rCRFR1-CHO media and lowest in ocCRFR1-CHO media without significant difference among the ocCRFR1-, moCRFR1-, and mbCRFR1-CHO, suggesting that the binding activities of oc-, mo-, mb-, and mCRFR1 with CRF differ among the plateau mammals, and mice, but not among the Tibetan's. While cAMP level stimulated by r/mUcn was highest in mCRFR1-CHO media and lowest in mbCRFR1-CHO media with significant difference among the plateau mammals, and mice, and also among the Tibetan's.We also found that water permeability mesured on CHO cells co-transfected rAQP4 with oc-,mb- or mo-CRFR1 stimulated by Ucn, but not CRF, are lower that with r-CRFR1. These results are in agreement with in vivo study, hypobaric hypoxia and CoCl2-hypoxia challenge induced diverse HPA axis responses, Increased CRF release from hypothalamus were well matched to enhanced cAMP levels in pituitary and plasma corticosterone/cortisol in all animals tested, and the responsivities in OC, MB and MO were low but high in mice.Hypoxia in vivo induced lower HPA axis responses in our plateau mammals than our lowland controls was well matched CRFRla differences lead to CRF/CRFR1α, cAMP production and water permeability measured on CHO cells transiently transfected with different CRFR1αs, suggesting that CRFRla sequences differences in plateau mammals is the reason of their low HPA axis responses to hypoxia.
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