Ghsr Cloning

The finding that the expression of the GHS-R was fully dependent on the addition of a single G protein subunit was unexpected since in previously published work the addition of a G protein subunit modulated an already existing activity. GHS-R expression could now be restored in oocytes obtained from >90% of the Xenopus frogs, suggesting that these oocytes did not contain sufficient quantities of Ga subunits to support GHS-R expression. This Xenopus GHS-R expression assay was utilized to screen pools of in vitro transcribed cRNAs derived from a swine pituitary cDNA library.

The authors evaluated the sensitivity of the assay by testing pools of cDNAs with a complexity of 10,000-20,000 cDNAs/pool for the presence of a GnRH-R- or TRH-R-derived signal (cloned receptors titrated in background cRNA). These receptors had been cloned earlier from tumor-derived poly (A)+ mRNA, which was enriched (~100-fold more receptor than native tissue) for the receptor of interest: GnRH, aT3-1 gonadotroph; thyrotropin-releasing hormone (TRH), Tt T mouse pituitary thyrotropic tumor) (14,15). Robust responses to TRH and GnRH could indeed be observed in the majority of eggs injected with a complex mixture of 10,000-20,000 individual cRNAs.

Screening for the presence of a cDNA that encoded the GHS-R in a swine pituitary cDNA library was initiated in pools with a complexity of 10,000 cRNAs. Following the evaluation of about 2 x 106 cRNAs, pool S10-20 gave a modest but reproducible bioluminescent response to challenge by MK-0677 (1 ^M). As shown in Fig. 3, isolation of a pure clone (7-3) resulted from the subfractionation of this pool of 10,000 cRNAs. Clone 7-3 conferred MK-0677-evoked bioluminescence in the aequorin assay, a large inward chloride current in oocytes (Fig. 3, bottom right panel), and high affinity binding of [35S]-MK-0677 to the GHS-R expressed in mammalian COS-7 cells (see Pharmological Characterization).

0 0

Post a comment