GPRASP2 maintains hematopoietic stem cell quiescence and self-renewal by receptor-mediated endocytosis

Abstract

Abstract The hematopoietic stem cell (HSC) compartment is highly heterogeneous in terms of functional diversity. Deeply quiescent HSCs, which are minimally active in homeostasis, exhibit superior repopulation potential upon transplantation. However, few tools exist to isolate these cells and interrogate mechanisms regulating the balance between quiescence and activation that maintains HSC fidelity. We previously reported that GPRASP2 (G-protein Coupled Receptor (GPCR)-associated Sorting Protein 2), which is critical for post-endocytic sorting to the lysosome of GPCRs, regulates cell-surface expression of C-X-C chemokine type receptor 4 on HSCs. GPRASP2 is mostly highly expressed in the HSCs relative to less primitive hematopoietic stem and progenitor cells, and our recent findings show GPRASP2 is also highly heterogeneous amongst single HSCs within both the human and mouse HSC compartment. Human/mouse HSCs enriched in GPRASP2 are transcriptionally programmed for stemness and quiescence programs. Here, we investigate whether GPRASP2 expression functionally marks and regulates the balance between HSC dormancy and activation. We generated Gprasp2-reporter mice to isolate Gprasp2high and Gprasp2low HSCs and perform serial (n>=12) and single-cell (n>=47) transplantation. Gprasp2highHSCs displayed slower repopulation kinetics, balanced reconstitution and increased, prolonged blood output compared to Gprasp2low HSCs.We crossed our Gprasp2-reporter model with a doxycycline-inducible H2B-GFP-expressing mouse model to track in vivo division kinetics and found that Gprasp2highHSCs rarely divide and retain GFP expression over a 150-day chase compared to Gprasp2low HSCs (n=6; p=0.0031). Further, ex vivo single-cell time-lapse imaging showing Gprasp2high HSCs are slower to divide, perform more symmetric divisions, and maintain higher levels of the stem marker SCA-1 in daughter cells (n>=277). Proteomic profiling on Gprasp2high/low HSCs reflected Gprasp2highHSCs are programmed for quiescence and also revealed enrichment in proteins relating to signaling and receptor-mediated endocytosis. Knockdown Gprasp2 in Gprasp2high/low HSCs revealed transcriptional changes predominantly in Gprasp2high HSCs, with upregulation of genes related to cell cycle, signaling, and receptor-mediated endocytosis. Transplantation of Gprasp2high/low HSCs after knockdown of Gprasp2 (n>=10)increased blood reconstitution of Gprasp2high HSCs, but revealed lower reconstitution of HSCs in the bone marrow, suggesting accelerated exhaustion. Ex vivo time-lapse imaging of single Gprasp2high HSCs (n>=167)after Gprasp2 knockdown revealed a shortened cell cycle (p=0.054), consistent with increased sensitivity to activation cues. Immunoprecipitation/mass spectrometry of GPRASP2 revealed engagement with endocytic trafficking proteins (SCYL-2, Clathrin heavy chain), further suggesting a regulatory role of receptor-mediated endocytosis. To test whether increased division kinetics were due to perturbed levels of receptor-mediated endocytosis, we assayed endocytic uptake and found that Gprasp2highHSCs have increased levels of endocytosis compared to Gprasp2lowHSCs (n=6; p=0.0032), and knockdown of Gprasp2 reduced endocytic uptake in Gprasp2highHSCs, but not Gprasp2low HSCs (n=5, p=0.0079). We specifically inhibited receptor-mediated endocytosis in ex vivo HSC expansion culture (n=12) and found that fewer HSCs expanded when either clathrin (chloropramazine; p=0.0326) or lysosome acidification (baflomycin; p=0.0078) was inhibited, suggesting endolysosomal degradation is important for HSC self-renewal. In total, these data reveal that a subset of HSCs rely on GPRASP2 to regulate receptor-mediated endocytosis to maintain quiescence and stemness. In conclusion, GPRASP2 marks a subset of deeply quiescent, highly transplantable HSCs. Mechanistically, increased GPRASP2 expression promotes HSC quiescence and stemness by enhancing receptor endocytosis and attenuating activating signals. These findings highlight a novel regulatory axis controlling HSC dormancy and suggest potential therapeutic targets for enhancing HSC activation during transplantation.