The constitutive activity of the adhesion GPCR GPR114/ADGRG5 is mediated by its tethered agonist.
Adhesion GPCRs (aGPCRs) form the second largest, yet most enigmatic class of the GPCR superfamily. Although the physiologic importance of aGPCRs was demonstrated in several studies, the majority of these receptors is still orphan with respect to their agonists and signal transduction. Recent studies reported that aGPCRs are activated through a tethered peptide agonist, coined the Stachel sequence. The Stachel sequence is the most C-terminal part of the highly conserved GPCR autoproteolysis-inducing domain. Here, we used cell culture-based assays to investigate 2 natural splice variants within the Stachel sequence of the orphan Gs coupling aGPCR GPR114/ADGRG5. There is 1 variant constitutively active in cAMP assays (∼25-fold over empty vector) and sensitive to mechano-activation. The other variant has low basal activity in cAMP assays (6-fold over empty vector) and is insensitive to mechano-activation. In-depth mutagenesis studies of these functional differences revealed that the N-terminal half of the Stachel sequence confers the agonistic activity, whereas the C-terminal part orientates the agonistic core sequence to the transmembrane domain. Sequence comparison and functional testing suggest that the proposed mechanism of Stachel-mediated activation is relevant not only to GPR114 but to aGPCRs in general.
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany.
Caroline
C
Wilde
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany.
Liane
L
Fischer
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany.
Vera
V
Lede
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany.
Jürgen
J
Kirchberger
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany.
Sven
S
Rothemund
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany.
Torsten
T
Schöneberg
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany ines.liebscher@medizin.uni-leipzig.de.
Ines
I
Liebscher
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation / Q000502:physiology*; D051379:Mice; D005810:Multigene Family; D009154:Mutation; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution
C000608294:GPR114 protein, mouse; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled
peptide agonist; receptor activation; receptor structure-function; signal transduction; tissue-specific splicing