Functional cross-interaction of the fragments produced by the cleavage of distinct adhesion G-protein-coupled receptors

J Biol Chem. 2009 Mar 6;284(10):6495-506. doi: 10.1074/jbc.M806979200. Epub 2009 Jan 5.

Abstract

The unusual adhesion G-protein-coupled receptors (aGPCRs) contain large extracellular N-terminal domains, which resemble cell-adhesion receptors, and C-terminal heptahelical domains, which may couple to G-proteins. These receptors are cleaved post-translationally between these domains into two fragments (NTF and CTF). Using the aGPCR latrophilin 1, we previously demonstrated that the fragments behave as independent cell-surface proteins. Upon binding the agonist, alpha-latrotoxin (LTX), latrophilin fragments reassemble and induce intracellular signaling. Our observations raised important questions: is the aGPCR signaling mediated by reassembled fragments or by any non-cleaved receptors? Also, can the fragments originating from distinct aGPCRs form hybrid complexes? To answer these questions, we created two types of chimerical constructs. One contained the CTF of latrophilin joined to the NTF of another aGPCR, EMR2; the resulting protein did not bind LTX but, similar to latrophilin, could couple to G-proteins. In another construct, the NTF of latrophilin was fused with the C terminus of neurexin; this chimera bound LTX but could not signal via G-proteins. Both constructs were efficiently cleaved in cells. When the two constructs were co-expressed, their fragments could cross-interact, as shown by immunoprecipitation. Furthermore, LTX(N4C) induced intracellular Ca2+ signaling only in cells expressing both constructs but not each individual construct. Finally, we demonstrated that fragments of unrelated aGPCRs can be cross-immunoprecipitated from live tissues. Thus, (i) aGPCR fragments behave as independent proteins, (ii) the complementary fragments from distinct aGPCRs can cross-interact, and (iii) these cross-complexes are functionally active. This unusual cross-assembly of aGPCR fragments could couple cell-surface interactions to multiple signaling pathways.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium Signaling / physiology*
  • Cell Adhesion / physiology
  • Cell Line, Tumor
  • Humans
  • Protein Structure, Tertiary / physiology
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins