# DEFINING RAPTOR-MEDIATED MECHANISMS OF HYPOXIC INJURY

> **NIH NIH R01** · UNIVERSITY OF WASHINGTON · 2024 · $639,640

## Abstract

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that is activated by nutrients and
energy, phosphorylating substrates to promote and coordinate anabolic metabolism. mTOR was identified
about thirty years ago in a yeast screen for mutants resistant to growth inhibition by the drug rapamycin; shortly
thereafter the highly conserved mammalian ortholog was discovered independently by biochemical methods.
Over the past thirty years, biochemical and genetic approaches have identified numerous evolutionarily
conserved components of the mTOR signaling pathway as well as the proteins forming the two distinct mTOR
complexes, mTORC1 and mTORC2. mTORC1 is composed of three core components, mTOR, Raptor, and
mLST8 (mammalian lethal with SEC13 protein 8); Rictor replaces Raptor in mTORC2. mTORC1 is inhibited by
rapamycin whereas mTORC2 is relatively insensitive. mTORC1 has been intensely studied because it
functions as a central regulator of the cell’s response to nutrients and energy and thereby impacts clinically
important conditions such as cancer, aging, and hypoxic injury. Through an unbiased mutant screen in C.
elegans for hypoxia resistant mutants, we have identified a missense partial-loss-of-function mutation in daf-
15, which encodes the sole C. elegans ortholog of Raptor (Ce-Raptor). A CRISPR/CAS9-generated mutant
with the identical lesion confirmed the mutation as conferring hypoxia resistance. Our discovery that reduction
of function of Ce-Raptor imparts resistance to hypoxic injury is consistent with published data using mTORC1
inhibitors in mammalian models. Our Ce-Raptor mutant called daf-15(gc67) is unique among published
metazoan Raptor mutants in that it is conditional; Raptor pathway functions can be turned off and on again by
simply varying temperature. daf-15(gc67) is essentially wild type at the standard culture temperature of 20°C,
hypoxia resistant at 22°C, and developmentally arrested at 25°C, the phenotype of daf-15 null mutants. The
graded temperature-conditional nature of the daf-15(gc67) phenotypes overcomes a critical barrier in the field
by providing a genetic reagent that allows temporal and tunable genetic control of Raptor and mTORC1
function for the first time in metazoans. This project will use this unique reagent to answer key questions about
how and when Raptor regulates hypoxic injury. In Aim 1, we will combine genetic and proteomic methods to
test specific hypotheses and discover the Raptor-regulated proteins that determine how and when Raptor
controls hypoxic injury. Using our conditional mutant, we have completed a screen for suppressors of Raptor
loss of function. In Aim 2, we will identify mutated genes suppressing Raptor loss-of-function and thereby
discover regulators of Raptor-mediated hypoxic sensitivity. Through these two aims, we will define how Raptor
regulates hypoxic sensitivity. Given the unbiased nature of the suppressor screen and proteomic studies, our
project has potential to id...

## Key facts

- **NIH application ID:** 10878881
- **Project number:** 5R01NS128769-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** C. Michael Crowder
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $639,640
- **Award type:** 5
- **Project period:** 2023-07-01 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10878881

## Citation

> US National Institutes of Health, RePORTER application 10878881, DEFINING RAPTOR-MEDIATED MECHANISMS OF HYPOXIC INJURY (5R01NS128769-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10878881. Licensed CC0.

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