Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-06
2001-09-18
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S091100, C435S091200, C336S020000, C336S020000, C336S020000
Reexamination Certificate
active
06291177
ABSTRACT:
BACKGROUND OF THE INVENTION
Communication between cells is essential to the maintenance of homeostasis of an organism. Extracellular signaling molecules, such as hormones and neurotransmitters, mediate cell-cell communication by acting through specific receptors located on the plasma membrane and in the cytoplasm of target cells.
G-protein coupled receptors are a class of seven-transmembrane domain polypeptides which transduce an extracellular signal into a cellular response. Following binding of a ligand to a G-protein coupled cell surface receptor, the G-protein coupled receptor activates an intracellular guanine nucleotide-binding protein, a G-protein, which mediates a cellular response to the extracellular signaling molecule (FIG.
1
).
G-proteins are heterotrimeric polypeptides composed of &agr;- , &bgr;- and &ggr;-subunits. Upon binding of ligand, the G-protein coupled receptor activates the G-protein by promoting the exchange of bound GDP for GTP in the &agr;-subunit and dissociation of the activated &agr;-subunit from &bgr;&ggr;-subunits (Lewin, B., “Signal Transduction”,
Genes VI,
Oxford University Press, New York, pp. 1053-1087 (1997)). The GTP-bound G&agr; subunit and the liberated &bgr;&ggr; dimeric subunit alter the activity of effectors in the target cell, for example, by altering the activity of adenylate cyclase and hence the levels of the second messenger cAMP, thereby altering the transcriptional activity of cAMP dependent genes.
G-proteins and G-protein-mediated cell signaling systems are highly conserved among eukaryotes from such diverse species as mammals, including humans, to yeast. (See, for example, Stryer, L. et al.,
Ann. Rev. Cell Biol.
2:391 (1986) and Lewin, B., “Signal Transduction”,
Genes VI,
Oxford University Press, New York, pp. 1053-1087 (1997)). Thus, due to the ease of experimental manipulation, it has been recognized that yeast can serve as a useful model for studying and evaluating G-protein coupled receptors and their ligands, as well as agents which act as antagonists and agonists of ligand activity in eukaryotic cells, including mammalian cells.
In the widely used yeast strain,
Saccharomyces cerevisiae,
two distinct G-protein &agr;-subunit proteins, Gpa1 and Gpa2, have been described (Miyajima, I., et al.,
Cell,
50:1011-1019 (1987); Nakafuku, M., et al.,
Proc. Natl. Acad. Sci. U.S.A.
85:1374-1378 (1988); Kübler, E., et al.,
J. Biol. Chem.
272:20321-20323 (1997)). Gpa1, also known as Scg1, is an &agr;-subunit of the heterotrimeric G-protein, which regulates a mitogen-activated protein kinase pathway that is required for the yeast response to mating pheromone. Gpa2 is the &agr;-subunit of a G-protein which is involved in pseudohyphal growth, and this G-protein functions by stimulating the yeast cAMP-response. The only known &bgr;- and &ggr;-subunits in yeast are Ste4 and Ste18, respectively (Whiteway, M., et al.,
Cell
56:467-477 (1989)).
To date, there have been reports of the use of Gpa1, the &agr;-subunit of a G-protein required to inhibit the pheromone response, with yeast and mammalian G-protein cell surface receptors, a process which results in Gpa1 activation upon ligand stimulation. For example, Gpa1 has been functionally linked to the rat A
2a
adenosine or human &bgr;2-adrenergic G-protein coupled receptor, resulting in adenosine agonist-dependent growth elicited by activation of the yeast pheromone-responsive pathway (Price, L. A., et al.,
Molec. Pharmacol.
50:829-837 (1996); Pausch, M. H., et al., U.S. Pat. No. 5,691,188 (1997)). Gpa1 has also been used with the rat somatostatin G-protein coupled receptor, resulting in growth-promoting signaling through pheromone-responsive pathways (Price, L. A., et al.,
Molec. Cell Biol.
15:6188-6195 (1995); Pausch, M. H., et al., U.S. Pat. No. 5,691,188 (1997)). Chimeric Gpal-mammalian G&agr; subunit proteins (Kang, Y. -S., et al.,
Molec. Cell Biol.
10:2582-2590 (1990); Price, L. A., et al.,
Molec. Cell Biol.
15:6188-6195 (1995); Medici, R., et al.,
EMBO J.
16:7241-7249 (1997)) and chimeric mammalian-yeast cell surface receptors (Pausch, M. H., et al., U.S. Pat. No. 5,691,188 (1997)) which bind ligands, have also been described. Mammalian G-protein coupled receptors have been functionally linked to mammalian G&agr; subunits through pheromone dependent pathways in yeast host cells lacking the endogenous GPA1 gene (King, K., et al., U.S. Pat. No. 5,482,835 (1996)).
These approaches require several genetic modifications of a typical laboratory yeast strain in order to effectively monitor the effects of extracellular signaling molecules, such as additional mutations in the FAR1 or SST2 genes. The existing technologies necessitate activation of the signal transduction pathway attributed to the a-subunit of the yeast G-protein, Gpa1, and are limited to evaluation based on pheromone-responsive mating criteria. Moreover, previous work has been limited to G-protein coupled receptors and G&agr; proteins which are normal cognate pairs (e.g., cell surface receptors and G-proteins which are able to associate and mediate effector pathways by G-protein activation). Additionally, these approaches often require deletion of the endogenous yeast G&agr; protein.
Thus, there is a continued need to develop new and improved methods for assessing agents which have agonistic and antagonistic effects on specific G-protein coupled receptors.
SUMMARY OF INVENTION
Work described herein shows that yeast cells transformed with a nucleic acid construct comprising a promoter operably linked to a first heterologous nucleic acid sequence encoding a G-protein coupled receptor which is operably linked to a second nucleic acid sequence encoding a G&agr; protein which is not a cognate protein of the mammalian G-protein coupled receptor, can be used to assess G-protein mediated signal transduction pathways. Expression of the first and second nucleic acid sequences produces a fusion protein in which the G&agr; protein is linked to the mammalian G-protein coupled receptor. Binding of a ligand to the mammalian G-protein coupled receptor activates the G&agr; protein, which in turn mediates a cellular response to the extracellular signal, such as regulation of specific effectors including adenylate cyclase and cyclic adenosine monophosphate (cAMP). In a particular embodiment the G&agr; protein is a yeast G&agr; protein, and in a particularly preferred embodiment the yeast G&agr; protein is Gpa2.
Thus, the invention relates to a transformed yeast cell comprising a nucleic acid construct comprising a promoter operably linked to a first heterologous nucleic acid sequence which is operably linked to a second nucleic acid sequence, wherein said first heterologous nucleic acid sequence encodes a mammalian G-protein coupled receptor, and wherein said second nucleic acid sequence encodes a G&agr; protein which is not a cognate protein of said G-protein coupled receptor, such that expression of the first and second DNA sequences produces a fusion protein wherein the G&agr; protein is linked to the mammalian G-protein coupled receptor. In one embodiment, binding of a ligand to the mammalian G-protein coupled receptor results in alteration of cellular levels or activity of an effector molecule (e.g., adenylate cyclase) or a second messenger (e.g., cAMP) or combinations thereof. In a preferred embodiment, the promoter is functional in yeast. In one embodiment, the G&agr; protein is a yeast G&agr; protein. In a preferred embodiment, the yeast G&agr; protein is Gpa2.
The invention also relates to a transformed yeast cell comprising a DNA construct comprising a promoter operably linked to a first nucleic acid sequence which is operably linked to a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a G-protein coupled receptor, and wherein the second nucleic acid sequence encodes a yeast Gpa2 protein, such that expression of the first and second nucleic acid sequences produces a fusion protein wherein the yeast Gpa2 protein is linked to the G-protein coupled receptor. In a particular embodiment, th
Errada Patrick R.
Gimeno Carlos J.
Madden Kevin T.
Fredman Jeffrey
Hamilton Brook Smith & Reynolds P.C.
Millennium Pharmaceuticals Inc.
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