A number of observations made in rodents as well as the phenotype of 46XY individuals harboring loss-of- or gain-of-function mutations of the lutropin receptor gene (LHR) clearly show that this receptor is important for the proliferation of the Leydig cells and may even be involved in the transformation of this cell type. The experiments proposed herein are designed to test the hypothesis that the LHR activates signaling cascades that promote the proliferation and/or survival of Leydig cells. Recent results from my laboratory have shown that two classic mitogenic/survival pathways, extracellular regulated kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt, are activated by the LHR in Leydig cells and that they are involved in their proliferation. We know virtually nothing about the mechanisms by which the LHR activates PI3K, but we have shown that the LHR-provoked activation of the ERK1/2 cascade involves two independent pathways. One requires the cAMP-activated protein kinase A (PKA) and the other requires Fyn, a Src-family kinase and the epidermal growth factor receptor (EGFR). We now propose to define the molecular basis of the LHR-induced activation of the ERK1/2 and PI3K/Akt cascades and to understand their roles in the proliferation, survival and differentiation of Leydig cells. The proposed experiments will be pursued using MA-10 Leydig tumor cells and primary cultures of immature rat Leydig cells and will be divided into five specific aims. (1) Define the mechanisms by which the LHR activates Fyn. (2) Complete the characterization of the involvement of EGF-like growth factors in the hCG- provoked ERK1/2 phosphorylation in Leydig cells (3) Define the mechanisms by which protein kinase A (PKA) mediates the LHR-provoked activation of the ERK1/2 cascade in Leydig cells; (4) Define the . involvement of the PI3K/Akt pathway in the proliferation and survival of Leydig cells and characterize the mechanisms by which the LHR activates this pathway. (5) Complete the characterization the functional consequences of the LHR-induced ERK1/2 and PI3K/Akt activation in Leydig cells. Some male reproductive disorders including feminization and precious puberty are associated with germ line or somatic mutations of the hLHR. In the testes the LHR is expressed exclusively in Leydig cells and these mutations not only cause disruptive endocrine manifestations but they also influence the number of Leydig cells, and can be associated with Leydig cell adenomas. Our studies are unique and novel in attempting to understand how does the LHR affect the proliferation of Leydig cells and thus to gain a better understanding of the pathophysiology of Leydig cell adenomas and disorders of sexual differentiation. ? ? ?
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