MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2
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BASIC RESEARCH www.jasn.org MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2 † ‡ † Michael J. Coady,* Abdulah El Tarazi, René Santer, Pierre Bissonnette, † † Louis J. Sasseville,* Joaquim Calado,§ Yoann Lussier, Christopher Dumayne, †| Daniel G. Bichet, and Jean-Yves Lapointe* *Physics Department & Groupe d’étude des protéines membranaires, †Departement of Molecular and Integrative Physiology & Groupe d’étude des protéines membranaires, and |Department of Medicine, Centre de recherche de l’Hôpital du Sacré-Cœur, University of Montreal, Montreal, Quebec, Canada; ‡Department of Pediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and §Department of Nephrology, ToxOmics, Centre for Toxicogenomics and Human Health, NOVA Medical School, New University of Lisbon, Lisbon, Portugal ABSTRACT The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa mem- BASIC RESEARCH brane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17– SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mu- tation (c.176+1G.A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as theA-kinaseanchorprotein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters. J Am Soc Nephrol 28: 85–93, 2017. doi: 10.1681/ASN.2015111282 Na+/glucose cotransporters employ the Na+ elec- SGLT2 mRNA, hindering characterization of this trochemical gradient to enable glucose uptake protein.6,7 against a concentration gradient. The low-affinity At least 11 pharmaceutical firms have candidate Na+/glucose cotransporter SGLT2, a product of the drugs for inhibiting SGLT2, including three that SLC5A2 gene (positioned at 16p11.2), is found al- are presently in clinical use, which should help most solely in the apical membranes of renal prox- imal tubules and reabsorbs over 90% of glucose from the glomerular filtrate.1 Although its cDNA Received November 30, 2015. Accepted April 5, 2016. was first cloned in 1992,2 the physiologic role of M.J.C. and A.E.T. contributed equally to this work. SGLT2 only became accepted a decade later follow- Published online ahead of print. Publication date available at ing identification of SLC5A2 mutations in a large www.jasn.org. majority of patients presenting with familial renal Correspondence: Dr. Jean-Yves Lapointe, Département de phy- 3–5 glucosuria (FRG). A major reason for this delay sique & Groupe d’étude des protéines membranaires, Université de was that, unlike the closely related SGLT1, SGLT2 Montréal, C.P. 6128, succ. Centre-ville, Montréal, Québec, Canada, does not express well either in transfected mamma- H3C 3J7. Email: [email protected] lian cells or in Xenopus laevis oocytes injected with Copyright © 2016 by the American Society of Nephrology J Am Soc Nephrol 28: 85–93, 2017 ISSN : 1046-6673/2801-85 85 BASIC RESEARCH www.jasn.org diabetic patients to control their glycemia by augmenting urinary glucose excretion.5 The drugs are all analogues of phlorizin (Pz), a specific inhibitor for the trans- porters of the SGLT family. Given the prev- alence of type 2 diabetes and the great potential for SGLT2 inhibitors in patients with this metabolic syndrome, it is possible that millions will be taking these drugs in the coming years. Our understanding of SGLT2, and of its inhibitors and their phys- iologic interactions, would obviously ben- efit from a robust expression system for Figure 1. Identification of MAP17 as a factor stimulating SGLT2-mediated AMG up- this protein. take. (A) The uptake of AMG into Xenopus oocytes expressing murine SGLT2 mRNA The SGLT2 protein had been shown to (4.6 ng/oocyte) was stimulated by coexpression of rat renal mRNA (46 ng/oocyte) (the 6 – be stable in oocytes and transfected cells four samples at left; mean SD; n=5 6 oocytes per sample). When the renal mRNA was size-fractionated, no transport activity was associated with expression of fractions even though it exhibited little transport – 8 B1 B4 (10 ng/oocyte) but these mRNA samples steeply increased mSGLT2 activity activity. This suggested the possibility when coinjected with mSGLT2 mRNA, proving that the factor responsible acted by that a second protein might be required augmenting the activity of the mSGLT2 protein. The Renal+mSGLT2 uptake was for SGLT2 to function and so we used ex- significantly different (P,0.001, ANOVA/Bonferroni) from the three other samples pression cloning to isolate the putative ac- shown on the left (Uninjected; Renal; mSGLT2). B3+mSGLT2 uptake is significantly cessory protein. This protein was identified different from mSGLT2 uptake (P,0.001). (B) Expression cloning using a cDNA library as MAP17, a 17 kDa subunit with 2 trans- produced from mRNA fraction B3 produced a single cDNA clone (rMAP17) whose membrane segments which had first been product very significantly augmented murine SGLT2 activity (P,0.001). cloned in 1995 as a protein whose tran- scription was upregulated in kidney, colon, breast, and lung cancers.9 oocytes with SGLT2 mRNA. Radiolabeled AMG uptake exper- iments were then performed to identify the pool of plasmids expressing the protein which complemented SGLT2. A plas- RESULTS mid from a single colony was eventually isolated, the tran- scribed product of which greatly stimulated SGLT2 activity Expression Cloning in oocytes (Figure 1B). The cDNA encoded by this clone was Expression cloning consists of coinjecting Xenopus oocytes fully sequenced and the transcribed protein was identified as with SGLT2 mRNA together with increasingly restricted sam- MAP17, product of the PDZK1IP1 gene. Similar results were ples of renal mRNA to ultimately identify a single protein seen when rat SGLT2 mRNA was coinjected with rat MAP17 that stimulates SGLT2 activity.10 Expression of either rat mRNA (data not shown). renal mRNA or mouse SGLT2 mRNA in oocytes led to en- hanced uptake of 14C-labeled a-methyl glucose (AMG, a Characterization of Human SGLT2–MAP17 Activity non-metabolized substrate for SGLT1/SGLT2), while SGLT2 We obtained human SGLT2 and MAP17 cDNAs by PCR coexpression with renal mRNA caused even greater uptake amplification and inserted them into pT7TS to enable tran- (Figure 1A). mRNA size-fractionation produced 24 fractions scription of polyA-tailed, capped mRNA. Coexpression with of renal mRNA, and aliquots were combined to create five human MAP17 in oocytes greatly stimulated human SGLT2– pooled samples (pools A– E). AMG uptakes with samples mediated AMG uptake (150620 fold for three experiments), from each pool indicated that pool B expressed the factor confirming that human MAP17 increases SGLT2 activity that affected AMG uptake. Subsequent oocyte injections of (Figure 2A). Na+/glucose cotransport generated currents of aliquots from the four size fractions contained in pool B are large amplitude (Figure 2B) which were not observed for con- shown in Figure 1A. The individual fractions did not induce a trol oocytes nor for oocytes solely expressing MAP17 or significant AMG uptake but coexpression of fractions B3 or B4 SGLT2. The cotransport current mediated by human SGLT2 with SGLT2 greatly stimulated AMG uptake. Thus, a protein was inhibited by Pz (a specific inhibitor which binds to the expressed by these size fractions of mRNA (approximately 0.5– glucose binding site) with a Ki of about 30 nM (data not 1.5 kb) augmented the level of SGLT2 activity. shown). Adding the high affinity inhibitor dapagliflozin at AcDNA library wasconstructed from mRNAsample B3and 10 nM in the presence of 2 mM glucose (Figure 2B) inhibited iterative screenings of pools of plasmids representing ever- the cotransport current by 90%, consistent with a Ki of 2.5 nM. smaller numbers of colonies were performed where mRNAwas Coexpression of MAP17 with other polyol-transporting mem- transcribed from the NotI-cut plasmids and coinjected into bers of the SLC5A family (i.e., SGLT1, SMIT1, SMIT2, SGLT3, 86 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 85–93, 2017 www.jasn.org BASIC RESEARCH examined, it can be seen that the sequence homology ends where the cytoplasmic C-terminal domains commence (Figure 2C). Coexpression of MARDI with SGLT2 showed that it stimulated SGLT2 activity by 1869fold,whichisquitesignificant butisalsoanorderofmagnitudeless than what is seen with MAP17 (Figure 2, AandD). Expression in Opossum Kidney Cells To better understand the MAP17–SGLT2 interaction, the two human cDNAs were separately inserted into the vector pcDNA3.1(-) and each received an epitope tag expected to face the extracellular solu- tion when