Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants

TY - JOUR

T1 - Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants

AU - Benned-Jensen, Tau

AU - Rosenkilde, M M

PY - 2010/3

Y1 - 2010/3

N2 - Background and purpose: In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms. Experimental approach: Expression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [ 35S]-GTPγS binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC 4 and LTD 4. Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively. Key results: The short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC 50. Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through Gα i. Conclusions and implications: We present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions.

AB - Background and purpose: In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms. Experimental approach: Expression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [ 35S]-GTPγS binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC 4 and LTD 4. Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively. Key results: The short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC 50. Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through Gα i. Conclusions and implications: We present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions.

KW - Binding, Competitive

KW - Brain

KW - Cell Line

KW - Gene Expression

KW - Humans

KW - Kidney

KW - Leukotrienes

KW - Ligands

KW - Myocardium

KW - Protein Binding

KW - Protein Isoforms

KW - Receptors, G-Protein-Coupled

KW - Reverse Transcriptase Polymerase Chain Reaction

U2 - 10.1111/j.1476-5381.2009.00633.x

DO - 10.1111/j.1476-5381.2009.00633.x

M3 - Journal article

C2 - 20148890

SN - 0007-1188

VL - 159

SP - 1092

EP - 1105

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

IS - 5

ER -