Protein Administration Restores Ghsr Coupling In Xenopus Oocytes

The expression of several receptors in heterologous cells is increased by the coexpres-sion of specific Ga subunits (8-13). To test whether G protein addition could increase the reliability of GHS-R expression the authors developed a Xenopus oocyte expression assay that incorporated the jellyfish photoprotein aequorin, which in the presence of calcium and the cofactor coelenterazine is chemi-luminescent. In these experiments aequorin protein is injected 2-3 d following poly (A)+ mRNA or library pool cRNA (12,13) injection into Xenopus eggs. The authors opted to coinject aequorin mRNA with pituitary poly (A)+ mRNA into oocytes. The use of aequorin mRNA obviated the need for a second injection resulting in lower background responses (20 vs 80 cps) and higher throughput. In addition, the aequorin mRNA provided a translational control for each oocyte. Coinjection of swine poly (A)+ mRNA with aequorin mRNA gave background light responses (30 cps) to MK-0677 when applied at a concentration of 1 ^M. However, when Ga11 cRNA was also coinjected (1:12 ratio [w/w] to poly (A)+ mRNA), robust light emission (~1000 cps) was evoked by 1 ^M MK-0677 (Fig. 2 and Table 1). MK-0677-induced bioluminescence is selectively dependent on Ga11 when expressed concurrently with swine poly (A)+ mRNA. MK-0677-stimulated bioluminescence could not be observed in the absence of poly (A)+ mRNA or when poly (A)+ mRNA was coinjected with six other individual Ga subunits given singly or in combination (PTX-sensitive: Gai1, Gai3, Gao; PTX-insensitive: Gaq, Ga13, Ga16) (Table 1). Positive responses could be recorded using either aequorin protein or aequorin mRNA (Fig. 2). Expression appeared maximal at 36-48 h postinjection, can be detected in as little as 18, and is attenuated by 72 h.

A frequently reported pitfall of oocyte expression systems is their inherent variability in expression of heterologous genes. Therefore, the authors tested oocytes from six different Xenopus frogs for their ability to express the GHS-R from the same batch of swine pituitary poly (A)+ mRNA. As shown in Table 2, all six frogs gave positive responses in almost all the eggs injected, dependent on the coexpression of Ga11. The magnitude of the bioluminescent response varied considerably, but did not prevent the assignment of a positive signal. To confirm and extend their initial observations, the authors evaluated the response of swine pituitary poly (A)+-injected oocytes to challenges with lower concentrations of MK-0677 and GHSs of diverse chemical structures, including peptides (GHRP-6, GHRP-2) and benzolactam GHSs (L-692,429 and its inactive enantiomer L-692,428). Bioluminescent responses could be observed for concentrations of MK-0677 as low as 1 nM (data not shown) whereas other bioactive GHSs elicited positive responses as well (Table 3). Additional tissues thought to contain GHS-Rs either by direct radioligand binding or by virtue of their in vivo biological response to GHSs were also evaluated. Poly (A)+ mRNA from human pituitary and rat hypothalamus and pituitary gave positive response to MK-0677, again strictly dependent on Ga11 expression (data not shown).

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