Interaction of the somatostatin receptor subtype 1 with the human homolog of the Shk1 kinase-binding protein from yeast.

Interaction between the C terminus of a G-protein-coupled receptor and intracellular constituents may represent a crucial step in regulating signal transduction. To identify potential interacting candidates the C terminus of the somatostatin receptor subtype 1 was used as bait in a yeast two hybrid screen of a human brain cDNA library. We identified the human Skb1 sequence (Skb1Hs) as interacting protein, which is homologous to the yeast protein known Skb1 to down-regulate mitosis in Schizosaccharomyces pombe via binding to the Shk1 protein kinase; the latter is a homolog to the mammalian p21(cdc42/Rac)-activated protein kinases. Interaction required almost the entire C terminus of the somatostatin receptor subtype 1 including the conserved NPXXY motif of transmembrane region seven; in the case of the Skb1Hs most of the N terminus and an S-adenosylmethionine binding domain were mandatory, whereas the C terminus was not essential. Interaction was verified by coexpression experiments in human embryonic kidney cells. As revealed by immunocytochemical analysis Skb1Hs expressed alone aggregates in large cytosolic clusters. When coexpressed, receptor subtype 1 and Skb1Hs were colocalized at the cell surface; these cells showed a strong increase in somatostatin binding compared with cells expressing the receptor only. This may suggest that Skb1Hs acts like a chaperone by correctly targeting the receptor to the cell surface.

Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1.

The Leydig insulin-like gene (Ley I-L), a member of the insulin-related gene family, is specifically expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. To determine the functional region of the mouse Ley I-L promoter and factors controlling the Ley I-L gene expression, we used 2.1 kb of the 5'-flanking region of the mouse Ley I-L gene to generate chimeric constructs with the chloramphenicol acetyltransferase gene (CAT). Transient transfections of MA10 Leydig cells, LTK- fibroblasts, and F9 embryonic cells by a series of 5'-deleted mouse Ley I-L promoter-CAT constructs revealed that the sequence between nucleotides -157 to +4 directs the transcription of the reporter gene in MA10 but not in LTK- and F9 cells, indicating that the determinants of Leydig cell-specific expression reside within this region. Deoxyribonuclease I (DNase I) footprint analysis revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase I-protected regions are homologous to the consensus binding site of the steroidogenic factor-1 (SF-1). Competition and antibody studies showed that the three SF-1-binding sites in the Ley I-L promoter have similar binding affinities for SF-1. Furthermore, transient transfections of MA10 cells with mutant reporter constructs, in which SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1-binding sites are required for SF-1-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing expression vector together with a Ley I-L promoter-CAT construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicated that SF-1 can transactivate the Ley I-L promoter. These data demonstrate an essential role of SF-1 in transcriptional activation of the Ley I-L promoter.