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Functional Characterization of Centrally Expressed Solute Carriers and G Protein-Coupled Receptors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transmembrane proteins are gatekeepers of the cells; controlling the transport of substrates as well as communicating signals among cells and between the organelles and cytosol. Solute carriers (SLC) and G protein-coupled receptors (GPCR) are the largest family of membrane transporters and membrane receptors respectively. The overall aim of this thesis was to provide a basic understanding of some of the novel SLCs and GPCRs with emphasis on expression, transport property, evolution and probable function.

The first part of the thesis directs towards the study of some novel solute carriers. In an initial study, we provided an overall picture of the sequence relationship and tissue expression of 14 diverse atypical SLCs confirming some of their evolutionary conservation and highly specific expression pattern. The focus then was on the SLC17 family (mainly vesicular proteins) and a novel member named Slc17a9. This study revealed that SLC17 family could be divided into four main phylogenetic clades which were all present before the divergence of the insect lineage with Slc17a9 having the most restricted evolutionary history. Detailed expression study of Slc17a9 in the mouse brain suggests that it is also expressed in some regions important for purinergic neurotransmission. Further, we deorphanised an aminoacid transporter Slc38a7 which was expressed in a majority of neurons in the CNS and showed that it preferably mediate transport of L–glutamine and L–histidine.

The second part of the thesis focuses on the study of two GPCRs belonging to the Rhodopsin superfamily, Gpr162 and Gpr153. A phylogenetic analysis revealed that both Gpr153 and Gpr162 originated from a common ancestor before the radiation of the mammalian lineage. Expression study revealed that Gpr162 had a predominant expression in the CNS and relatively lower expression in the other tissue tested whereas Gpr153 had a more widespread and similar expression pattern in both CNS and peripheral tissues. The functional studies of the two GPCRs were done using the antisense oligodeoxynucleotide knockdown rat model. These studies provided evidence linking the orphan Gpr162 gene with the regulation of food intake– related behaviour whereas Gpr153 gene caused only a slight reduction in food intake.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 689
Keyword [en]
GPCR, SLC, Gpr153, Gpr162, Slc17, Slc38
National Category
Neurosciences
Research subject
Neuroscience
Identifiers
URN: urn:nbn:se:uu:diva-156832ISBN: 978-91-554-8120-9 (print)OAI: oai:DiVA.org:uu-156832DiVA: diva2:433405
Public defence
2011-09-22, B42, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-09-01 Created: 2011-08-09 Last updated: 2011-11-03Bibliographically approved
List of papers
1. Long evolutionary conservation and considerable tissue specificity of several atypical solute carrier transporters
Open this publication in new window or tab >>Long evolutionary conservation and considerable tissue specificity of several atypical solute carrier transporters
2011 (English)In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 478, no 1-2, 11-18 p.Article in journal (Refereed) Published
Abstract [en]

The superfamily of Solute Carriers (SLCs) has around 384 members in the human genome grouped into at least 48 families. While many of these transporters have been well characterized with established important biological functions, there are few recently identified genes that are not studied regarding tissue distribution or evolutionary origin. Here we study 14 of these recently discovered SLC genes (HIAT1, HIATL1, MFSD1, MFSD5, MFSD6, MFSD9, MFSD10, SLC7A14, SLC7A15, SLC10A6, SLC15A5, SLC16A12, SLC30A10 and SLC21A21) with the purpose to give much better picture over the sequence relationship and tissue expression of the diverse SLC gene family. We used a range of bioinformatic methods to classify each of these genes into the different SLC gene families. We found that 9 of the 14 atypical SLCs are distant members of the Major Facilitator Superfamily (MFS) clan while the others belong to the APC clan, the DMT clan, the CPA_AT clan and the IT clan. We found most of the genes to be highly evolutionary conserved, likely to be present in most bilateral species, except for SLC21A21 that we found only present in mammals. Several of these transporter genes have highly specific tissue expression profile while it is notable that most are expressed in the CNS with the exception of SLC21A21 and SLC15A5. This work provides fundamental information on 14 transporters that previously have not received much attention enabling a more comprehensive view over the SLC superfamily.

Keyword
APC, CNS, Kidney, Liver, MFS, Transport
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-135048 (URN)10.1016/j.gene.2010.10.011 (DOI)000290888100002 ()21044875 (PubMedID)
Available from: 2010-12-03 Created: 2010-12-03 Last updated: 2017-12-12Bibliographically approved
2. Glutamate, aspartate and nucleotide transporters in the SLC17 family form four main phylogenetic clusters: evolution and tissue expression
Open this publication in new window or tab >>Glutamate, aspartate and nucleotide transporters in the SLC17 family form four main phylogenetic clusters: evolution and tissue expression
Show others...
2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, no 1, 17- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The SLC17 family of transporters transports the amino acids: glutamate and aspartate, and, as shown recently, also nucleotides. Vesicular glutamate transporters are found in distinct species, such as C. elegans, but the evolutionary origin of most of the genes in this family has been obscure. RESULTS: Our phylogenetic analysis shows that the SLC17 family consists of four main phylogenetic clades which were all present before the divergence of the insect lineage. One of these clades has not been previously described and it is not found in vertebrates. The clade containing Slc17a9 had the most restricted evolutionary history with only one member in most species. We detected expression of Slc17a1-17a4 only in the peripheral tissues but not in the CNS, while Slc17a5- Slc17a9 are highly expressed in both the CNS and periphery. CONCLUSIONS: The in situ hybridization studies on vesicular nucleotide transporter revealed high expression throughout the cerebral cortex, certain areas in the hippocampus and in specific nuclei of the hypothalamus and thalamus. Some of the regions with high expression, such as the medial habenula and the dentate gyrus of the hippocampus, are important sites for purinergic neurotransmission. Noteworthy, other areas relying on purine-mediated signaling, such as the molecular layer of the dentate gyrus and the periaqueductal gray, lack or have a very low expression of Slc17a9, suggesting that there could be another nucleotide transporter in these regions.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-135068 (URN)10.1186/1471-2164-11-17 (DOI)000275279400001 ()20059771 (PubMedID)
Available from: 2010-12-03 Created: 2010-12-03 Last updated: 2017-12-12Bibliographically approved
3. Identification of SLC38A7 (SNAT7) Protein as a Glutamine Transporter Expressed in Neurons
Open this publication in new window or tab >>Identification of SLC38A7 (SNAT7) Protein as a Glutamine Transporter Expressed in Neurons
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 23, 20500-20511 p.Article in journal (Refereed) Published
Abstract [en]

The SLC38 family of transporters has in total 11 members in humans and they encode amino acid transporters called sodium-coupled amino acid transporters (SNAT). To date, five SNATs have been characterized and functionally subdivided into systems A (SLC38A1, SLC38A2, and SLC38A4) and N (SLC38A3 and SLC38A5) showing the highest transport for glutamine and alanine. Here we present identification of a novel glutamine transporter encoded by the Slc38a7 gene, which we propose should be named SNAT7. This transporter has L-glutamine as the preferred substrate but also transports other amino acids with polar side chains, as well as L-histidine and L-alanine. The expression pattern and substrate profile for SLC38A7 shows highest similarity to the known system N transporters. Therefore, we propose that SLC38A7 is a novel member of this system. We used in situ hybridization and immunohistochemistry with a custom-made antibody to show that SLC38A7 is expressed in all neurons, but not in astrocytes, in the mouse brain. SLC38A7 is unique in being the first system N transporter expressed in GABAergic and also other neurons. The preferred substrate and axonal localization of SLC38A7 close to the synaptic cleft indicates that SLC38A7 could have an important function for the reuptake and recycling of glutamate.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-155238 (URN)10.1074/jbc.M110.162404 (DOI)000291267600037 ()
Available from: 2011-06-21 Created: 2011-06-20 Last updated: 2017-12-11Bibliographically approved
4. GPR162 is expressed in the hypothalamus and is involved in food intake related behaviour
Open this publication in new window or tab >>GPR162 is expressed in the hypothalamus and is involved in food intake related behaviour
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2011 (English)Article in journal (Other academic) Submitted
Abstract [en]

The Rhodopsin family of G protein-coupled receptors (GPCRs) includes about 270 non-olfactory receptors and is the largest family of GPCRs. About sixty non-olfactory Rhodopsin GPCRs are still orphans without known ligands, and fairly little is known about their functions. In this study, we present molecular, neuroanatomical, genetic and behavioral data implicating a Rhodopsin family protein, GPR162, in the regulation of food intake-related behaviour and glucose homeostasis. The real-time PCR data show that GPR162 is predominantly expressed in the CNS. The in situ hybridization results confirmed significant expression of GPR162 in several hypothalamic sites, amygdala, substantia nigra and ventral tegmental area, among others regions. In line with the distribution of the GPR162 mRNA in the feeding circuitry, antisense oligo knockdown of GPR162 caused a significant reduction in food intake but no effect was observed towards reduction in body weight in rats. Our human genetics studies suggest that genetic variants of GPR162 affect glucose homeostasis. In conclusion, this study provides evidence linking the orphan GPR162 gene with the regulation of food intake-related behaviour.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156816 (URN)
Available from: 2011-08-09 Created: 2011-08-09 Last updated: 2011-12-22Bibliographically approved
5. The G protein coupled receptor Gpr153 shares common evolutionary origin with Gpr162 and is highly expressed in central regions including the thalamus, cerebellum and the arcuate nucleus
Open this publication in new window or tab >>The G protein coupled receptor Gpr153 shares common evolutionary origin with Gpr162 and is highly expressed in central regions including the thalamus, cerebellum and the arcuate nucleus
Show others...
2011 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 24, 4881-4894 p.Article in journal (Refereed) Published
Abstract [en]

The Rhodopsin family of G protein-coupled receptors (GPCRs) includes the phylogenetic α-group consisting of about 100 human members. The α-group is the only group of GPCRs that has many receptors for biogenic amines which are major drug targets. Several members of this group are orphan receptors and their functions are elusive. In this study we present a detailed phylogenetic and anatomical characterization of the Gpr153 receptor and also attempted to study its functional role. We identified the homologue of GPR153 in the elephant shark genome and phylogenetic and synteny analyses revealed that Gpr162 originated from Gpr153, through a duplication event before the radiation of the amphibian lineage. Quantitative real time PCR study reveals wide spread expression of GPR153 in the CNS and all the peripheral tissues investigated. Detailed in situ hybridization on mouse brain showed specifically high expression in the thalamus, cerebellum and the arcuate nucleus. The antisense oligodeoxynucleotide knockdown of GPR153 caused a slight reduction in food intake and the elevated plus maze test showed significant reduction in the percentage of time spent in the centre square, which points towards a probable role in decision making. This report provides the first detailed characterization of the evolution, expression and as well as primary functional properties of the GPR153 gene.

Keyword
GPCR, GPR153, GPR162, food intake, anxiety, decision making
National Category
Neurosciences
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-156817 (URN)10.1111/j.1742-4658.2011.08388.x (DOI)000297737500015 ()21981325 (PubMedID)
Available from: 2011-08-09 Created: 2011-08-09 Last updated: 2017-12-08Bibliographically approved

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