ABSTRACT
Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin and calcitonin (CT) are structurally and functionally related neuropeptides. It has recently been shown that the molecular pharmacology of CGRP and ADM is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Furthermore, RAMP proteins have also been shown to govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. In this study, we have cloned the rat RAMP family and characterized the pharmacology of rat CGRP and ADM receptors. Rat RAMP1, RAMP2 and RAMP3 shared 72%, 69% and 85% homology with their respective human homologues. As expected CRLR-RAMP1 coexpression conferred sensitivity to CGRP, whilst association of RAMP2 or RAMP3 with CRLR conferred high affinity ADM binding. Using specific oligonucleotides we have determined the expression of RAMP1, RAMP2 and RAMP3 mRNAs in the rat central nervous system by in situ hybridization. The localization of RAMP mRNAs was heterogeneous. RAMP1 mRNA was predominantly expressed in cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most abundant in hypothalamus; and RAMP3 was restrictively expressed in thalamic nuclei. Interestingly, in specific brain areas only a single RAMP mRNA was often detected, suggesting mutual exclusivity in expression. These data allow predictions to be made of where each RAMP protein may heterodimerize with its partner G-protein-coupled receptor(s) at the cellular level and consequently advance current understanding of cellular sites of action of CGRP, ADM, amylin and CT. Furthermore, these localization data suggest that the RAMP family may associate and modify the behaviour of other, as yet unidentified neurotransmitter receptors.
Subject(s)
Central Nervous System/metabolism , Membrane Proteins/genetics , Neuropeptides/metabolism , RNA, Messenger/metabolism , Receptors, Calcitonin/metabolism , Adrenomedullin , Amino Acid Sequence/physiology , Amyloid/metabolism , Animals , Calcitonin/metabolism , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Receptor-Like Protein , Cells, Cultured , Cloning, Molecular , DNA, Complementary/isolation & purification , Diencephalon/metabolism , Intracellular Signaling Peptides and Proteins , Islet Amyloid Polypeptide , Male , Membrane Proteins/metabolism , Mesencephalon/metabolism , Molecular Sequence Data , Peptides/metabolism , Rats , Rats, Sprague-Dawley , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Protein 2 , Receptor Activity-Modifying Protein 3 , Receptor Activity-Modifying Proteins , Rhombencephalon/metabolism , Sequence Homology, Amino Acid , Spinal Cord/metabolism , Telencephalon/metabolismABSTRACT
The murine Na(+)/myo-inositol cotransporter (SLC5A3) gene (Slc5a3) was cloned, the restriction sites mapped, and the coding region sequenced. Similar to other mammalian counterparts, including human, the gene has a single coding exon, with an open reading frame of 2.2 kb. The predicted protein of 718 amino acids is also highly conserved, compared to other mammalian homologs. Using fluorescence in situ hybridization, Slc5a3 was localized to the telomeric region of mouse chromosome 16, which is syntenic to human chromosome 21q22. An increased Slc5a3 copy number may explain the increased levels of myo-inositol in the brains of trisomy 16 mice and the increased rate of transport of myo-inositol into cultured neurons derived from trisomy 16 mice.
Subject(s)
Carrier Proteins/genetics , Chromosomes, Human, Pair 21/genetics , Heat-Shock Proteins/genetics , Membrane Proteins , Physical Chromosome Mapping , Symporters , Telomere/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Down Syndrome/genetics , Exons/genetics , Humans , In Situ Hybridization, Fluorescence , Mice , Molecular Sequence Data , Open Reading Frames/genetics , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The genomic structure, transcription start site, polyadenylation signals, and promoter of the human Na+/ myo-inositol cotransporter (SLC5A3) gene have been elucidated through cloning, sequencing, mRNA analyses, and reporter gene assays. The gene consists of one promoter and two exons spanning approximately 26 kb. Exon 1 contains 175 bp of 5' untranslated sequence and is 15 kb upstream of exon 2. The 9.5-kb exon 2 contains the entire 2157-bp open reading frame and a large 3' untranslated sequence with seven putative polyadenylation signals. Multiple messages with different-sized 3' untranslated regions can be detected on Northern blots. Hypertonic stress caused mRNA levels, and primarily that of the full-length 9.5-kb transcript, to increase in cultured melanoma cells; ribonuclease protection analysis demonstrated that the transcription start site was the same in stressed as in control cells. The SLC5A3 gene functions in cellular osmoregulation and is expressed in many human tissues including the brain, kidney, and placenta. It is localized to chromosome 21q22.1. An overexpression of the SLC5A3 gene deserves consideration as a factor in the pathophysiology of Down syndrome.
Subject(s)
Carrier Proteins/genetics , Heat-Shock Proteins/genetics , Membrane Proteins , Promoter Regions, Genetic , Symporters , Animals , Base Sequence , Binding Sites , Cell Line , Dogs , Humans , Molecular Sequence Data , Peptide Chain Initiation, Translational , Protein Biosynthesis , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
The human osmoregulatory Na+/myo-inositol cotransporter gene (SLC5A3) was recently cloned and localized to the region of 21q22. Fine mapping of this gene was accomplished by identifying YAC clones that contain SLC5A3 and utilizing known STS markers for 21q22.1 and 21q22.2 sub-bands that map to the positive YAC clones. Two bacteriophage P1 clones containing the SLC5A3 gene gave a positive PCR product when screened with the 21q22.1 marker VN02, an expressed sequence tag (EST00541). Through DNA sequence analysis, it was determined that this STS marker if part of the 3' untranslated region of the SLC5A3 gene.
Subject(s)
Carrier Proteins/genetics , Chromosomes, Human, Pair 21/genetics , Genetic Markers , Heat-Shock Proteins/genetics , Membrane Proteins , Symporters , Bacteriophages/genetics , Base Sequence , DNA Primers , DNA, Complementary/genetics , Gene Expression , Genes , Humans , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
A human Na+/myo-inositol cotransporter (SLC5A3) gene was cloned; sequencing revealed a single intron-free open reading frame of 2157 nucleotides. Containing 718 amino acid residues, the predicted protein is highly homologous to the product of the canine osmoregulatory SLC5A3 gene. The SLC5A3 protein is number 3 of the solute carrier family 5 and was previously designated SMIT. Using fluorescence in situ hybridization, the human SLC5A3 gene was localized to band q22 on chromosome 21. Many tissues including brain demonstrate gene expression. The inability of a trisomic 21 cell to downregulate expression of three copies of this osmoregulatory gene could result in increased flux of both myo-inositol and Na+ across the plasma membrane. The potential consequences include perturbations in the cell membrane potential and tissue osmolyte levels. The SLC5A3 gene may play a role in the pathogenesis of Down syndrome.
