Neuropilin 1 (NP1) is a receptor for both semaphorin and vascular endothelial growth factor expressed by subpopulations of neuronal and endothelial cells. In the immune system, NP1 is present on dendritic and regulatory T cells. Here, we show that NP1 is expressed in the murine thymus, starting on day 12.5 of gestation. In the adult, NP1 is mainly expressed by CD4(-)CD8(-) double negative cells, CD4+CD8+ double positive cells, and CD4+CD25+ regulatory T cells but barely detected in single CD4+ and CD8+ positive thymocytes. Within the CD4(-)CD8(-)CD3(-) (triple-negative, TN) immature cells, NP1 expression starts in TN3 (CD44(-)CD25+) and increases in TN4 (CD44(-)CD25(-)) cells. In order to study the role of NP1 in thymocyte differentiation, we generated mice in which the np1 gene is selectively disrupted in the T-cell lineage. The mutant mice display normal thymocyte, peripheral, conventional and CD4+CD25+Foxp3+ regulatory T-cell populations. However, we observe a down-regulation of the CD25 expression between the TN3 and TN4 stages that is (i) correlated to increased expression of NP1 in control mice and (ii) altered in mutant mice, suggesting that NP1 is co-regulated with CD25 during early immature thymocyte differentiation.

We have recently shown that a heterotrimeric G i protein is coupled to the erythropoietin (Epo) receptor. The G i protein constitutively associates in its heterotrimeric form with the intracellular domain of Epo receptor (EpoR). After Epo stimulation G i is released from the receptor and activated. In the present study we have investigated the functional role of the heterotrimeric G i protein bound to EpoR. In Chinese hamster ovary cells expressing EpoR, the G i inhibitor pertussis toxin blocked mitogen-activated protein kinase (MAPK) Erk1/2 activation induced by Epo. Epo-dependent MAPK activation was also sensitive to the G␤␥ competitive inhibitor ␤ARK1-ct (C-terminal fragment of the ␤-adrenergic receptor kinase), to the Ras dominant negative mutant RasN17, and to the phosphoinositide 3-kinase (PI3K) inhibitor LY 294002. A region of 7 amino acids (469 -475) in the C-terminal end of EpoR was shown to be required for G i binding to EpoR in vivo. Deletion of this region in EpoR abolished both MAPK and PI3K activation in response to Epo. We conclude that in Chinese hamster ovary cells, Epo activates MAPK via a novel pathway dependant on G i association to EpoR, G␤␥ subunit, Ras, and PI3K. The tyrosine kinase Jak2 also contributes to this new pathway, more likely downstream of ␤␥ and upstream of Ras and PI3K. This pathway is similar to the best characterized pathway used by seven transmembrane receptors coupled to G i to activate MAPK and may cooperate with other described Epo-dependent MAPK activation pathways in hematopoietic cells.