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Yale University School of Medicine, Department of Cellular and Molecular Physiology, New Haven, Connecticut, USA
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Yale University School of Medicine, Department of Cellular and Molecular Physiology, New Haven, Connecticut, USA
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Yale University School of Medicine, Department of Cellular and Molecular Physiology, New Haven, Connecticut, USA
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Yale University School of Medicine, Department of Cellular and Molecular Physiology, New Haven, Connecticut, USA
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The steroidogenic acute regulatory protein (StAR) is critical to the regulated synthesis of steroids in vertebrates. We have isolated cDNA sequences encoding StAR in the freshwater stingrays Potamotrygon hystrix and P. motoro. A single P. hystrix StAR transcript (3376 bp) and two overlapping P. motoro StAR transcripts (1272 and 3365 bp) were isolated. The P. hystrix and P. motoro StAR transcripts contain open reading frames encoding proteins of 284 amino acids that are 99% identical to each other and 56–64% identical to other StAR proteins. Pregnenolone synthesis by green monkey kidney (COS-1) cells transfected with an expression construct encoding a human cholesterol side chain cleavage/adrenodoxin reductase/adrenodoxin fusion protein was increased 16-fold by coexpression with a pCMV5/P. motoro StAR expression construct. Northern blot analysis revealed a single 4000 bp StAR transcript in the P. motoro interrenal gland, but RT-PCR indicates StAR mRNA is also expressed in the brain, gonads, atria, ventricle, gill (female only) and muscle (female only). Expression in extragonadal and extraadrenocortical tissues is an indication that StAR may be critical to processes other than steroidogenesis. The longest P. motoro StAR transcript contains a sequence with great similarity to short interspersed repetitive elements found in other elasmobranchs. This study is the first to isolate and characterize elasmobranch StAR cDNA sequences and to demonstrate the activity of a nonmammalian StAR protein in a heterologous expression system.
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ABSTRACT
The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, hαCTR and hβCTR. The two cDNAs are identical except that the hαCTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (hαCTR Kd 0·09 nm, hβCTR Kd 0·12 nm). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the hβCTR isoform was expressed at tenfold higher levels than the hαCTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in β-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.
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Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia
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Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria, Australia
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Department of Pharmacology, Monash University, Clayton, Victoria, Australia
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Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and in situ hybridisation that Insl5 mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr202/Tyr204), AKT (Thr308 and Ser473) and S6RP (Ser235/236) and inhibited cAMP production but did not stimulate Ca2+ release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing RXFP4. Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.
