Methods relating to mammalian raptor polypeptide
The present invention relates to isolated raptor nucleic acid molecules of mammalian origin (e.g., human) and complements, portions and variants thereof. Another aspect of the invention are isolated raptor polypeptides of mammalian origin and portions thereof, and antibodies or antigen binding fragments thereof that specifically bind a raptor polypeptide. The present invention also relates to constructs and host cells comprising the nucleic acid molecules described herein. In addition, the present invention relates to uses of the nucleic acid and polypeptide molecules provided herein.
1. A method of isolating a mammalian raptor polypeptide in a sample and assessing interaction of the mammalian raptor polypeptide with mTOR comprising:
a) contacting the sample with an antibody or antigen binding fragment thereof that specifically binds to a polypeptide encoded by SEQ ID NO: 1;
b) isolating the polypeptide in the sample which specifically binds to the antibody or antigen binding fragment thereof, thereby isolating a mammalian raptor polypeptide; and
c) assessing interaction of mTOR with the mammalian raptor polypeptide isolated in step b), wherein said assessing comprises detecting an mTOR-mammalian raptor polypeptide complex, thereby assessing interaction of the mammalian raptor polypeptide with mTOR.
2. The method of claim 1 , wherein said assessing interaction of mTOR with the mammalian raptor polypeptide isolated in step b) comprises assaying kinase activity.
3. The method of claim 1 , wherein said assessing interaction of mTOR with the mammalian raptor polypeptide isolated in step b) comprises detecting mTOR after dissociation of the complex.
4. A method of identifying an agent that alters binding of a mammalian raptor protein with mammalian target of rapamycin (mTOR) protein comprising:
a) contacting a polypeptide whose amino acid sequence is at least 95% identical to SEQ ID NO:2 with mTOR protein under conditions in which the raptor protein interacts with the mTOR protein, with an agent to be assessed;
b) assessing the extent to which raptor binds with mTOR in the presence of the agent to be assessed, wherein said assessing comprises detecting mTOR associated with the polypeptide; and
c) comparing the level of mTOR-mammalian raptor protein complex in the presence of the agent with the level in the absence of the agent; wherein a difference in the level indicates the agent alters the binding of a mammalian raptor protein with mTOR protein.
5. A method of identifying an agent that alters the interaction of raptor polypeptide with mTOR, the method comprising (a) contacting a mammalian cell lysate that comprises a polypeptide whose amino acid sequence is at least 95% identical to SEQ ID NO:2 with an antibody or antibody fragment that specifically binds the polypeptide of SEQ ID NO:2, wherein the mammalian cell lysate has been prepared under conditions appropriate to preserve interaction between the polypeptide of SEQ ID NO: 2 and mTOR, and wherein the mammalian cell lysate has been prepared from cells that have been contacted with a test compound; (b) isolating material bound to the antibody or antibody fragment from the mammalian cell lysate; and (c) analyzing the isolated material for the presence or activity of the mTOR-raptor protein complex, wherein an alteration in the presence or activity of the mTOR-raptor protein complex indicates that the test compound is an agent that alters interaction of raptor polypeptide with mTOR.
6. The method of claim 5 , wherein the polypeptide is a recombinant polypeptide comprising a tag, and the antibody or antibody fragment binds to the tag.
7. The method of claim 5 , wherein analyzing the mTOR-raptor protein complex comprises assaying mTOR kinase activity of the complex.
8. The method of claim 5 , wherein said analyzing the isolated material for the presence of activity of the mTOR-raptor protein complex comprises detecting mTOR after dissociation of the complex.
9. The method of claim 5 , wherein the polypeptide in step (a) comprises the amino acid sequence of SEQ ID NO:2 step (a) comprises SEQ ID NO:2.
10. The method of claim 9 , wherein analyzing the mTOR-raptor protein complex comprises assaying mTOR kinase activity of the complex.