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In order to demonstrate the potential of the LCR/MEL expression system for the expression of seven-transmembrane helix receptors the human calcitonin receptor (hCTR) has been cloned and expressed at high levels in this system. Using the newly developed single-step expression vectors pEV and pNV, stable recombinant MEL cells which express the hCTR at ~6 pmol of receptor/mg of total cell protein (60 pmol/mg of membrane protein), or 1.9 × 106 receptors per cell, have been generated. Ligand binding studies have shown that the cloned receptor expressed in MEL cells has properties which are indistinguishable from those of the native receptor in T47D cells. It is suggested that MEL cells expressing seven-transmembrane helix receptors provide an efficient and readily attainable source of material for ligand binding experiments using these receptors.Copyright 1995, 1999 Academic Press, Inc.

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, hαCTR and hβCTR. The two cDNAs are identical except that the hαCTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (hαCTR Kd0·09 nm, hβCTR Kd0·12 nm). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the hβCTR isoform was expressed at tenfold higher levels than the hαCTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in β-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.