Zero-length cross-linking of the LH receptor reveals tight interactions between extracellular and transmembrane domains during receptor activation

Abstract

Luteinizing hormone (LH) secreted by the anterior pituitary is critical for normal sexual development and function. Activation of the LH receptor in target tissues involves LH binding to the extracellular domain (ECD), thereby modifying interactions between the ECD and transmembrane domain (TMD). To analyse these intramolecular changes we developed a recombinant LH/CG receptor (LHR) bearing an intramolecular cleavage site at position 316 recognized by the proprotein convertase furin (LHR-F316). Western blot studies indicated normal synthesis, glycosylation and expression of the recombinant LHR with complete cleavage into both domains, ECD-316 and 316-TMD. Affinity binding for hCG as well as basal and hormone induced cAMP production were identical to the wildtype LHR, indicating intact functional activity of the cleaved LHR. Physical interaction between the ECD and TMD was studied by zero-length cross-linking on HEK 293 cells expressing LHR-F316. No difference in cross-linking intensity between both domains was observed after ligand binding or in constitutively active receptors. Incubation of cells expressing LHR-F316 with dithiotreitol led to a shedding of the ECD-316 from the 316-TMD without increase of the basal adenylate cyclase activity. Our results show that activation of the LH receptor does not result from a switch of an inverse agonist effect of the ECD-316 to an agonist effect but is related to subtle modifications of intramolecular interactions between ECD and TMD.