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Monocyte chemoattractant protein-1 (MCP-1) is a member of the Cys-Cys chemokine family. Two related MCP-1 receptors have been identified (CC-CKR2A and CC-CKR2B), although the precise kinetics of ligand binding and calcium signaling of these receptors has yet to be investigated. To examine this more closely, the human MCP-1 receptors were cloned and expressed in Chinese hamster ovary (CHO) cells. Membranes prepared from cells expressing CC-CKR2B bind MCP-1 selectively and with high affinity (Kd = 120 pM). MCP-1 stimulation of recombinant CHO cells expressing CC-CKR2B induces a rapid increase in intracellular Ca2+ through both receptor-operated Ca2+ channels and mobilization of Ca2+ from intracellular stores, and leads to a rapid temperature-dependent internalization of the ligand/receptor complexes. In contrast, recombinant CHO cells expressing CC-CKR2A, and membranes prepared from these cells, fail to bind detectable levels of MCP-1. However, MCP-1 stimulation of cells expressing CC-CKR2A induces a small but significant increase in intracellular Ca2+. Repeated stimulation of these cells with MCP-1 leads to a potentiation of the response to a level comparable to that seen in cells expressing CC-CKR2B. These observations suggest that the levels of cell surface CC-CKR2A are controlled by novel mechanisms.

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, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR 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 (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 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 h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.