Stable expression of the recombinant human VIP1 receptor in clonal Chinese hamster ovary cells: Pharmacological, functional and molecular properties

We stably transfected Chinese hamster ovary (CHO) cells with the recombinant human vasoactive intestinal peptide (VIP), receptor. A clone referred to as Clone 15 was isolated and studied for receptor properties. The following data were obtained: (1) one class of binding site was identified by Scatchard analysis of [I-125]VIP binding to cell membranes with a K-d of 0.41 nM and a B-max pmol/mg protein; (2) the constant K-i for the inhibition of [I-125]VIP binding by VIP and related peptides was: VIP (0.9 nM) = pituitary adenylate cyclase-activating peptide (PACAP)-27 (1.3 nM) < PACAP-38 (6.8 nM) < helodermin (46.0 nM) < human growth hormone-releasing factor (GRF) (0.6 mu M) < peptide histidine methionineamide (2.0 mu M) < secretin (> 10 mu M); (3) cross-linking experiments using [I-125]VIP identified a single M-r 67 000 recombinant receptor; (4) VIP stimulated cAMP production in Clone 15 cells an EC50 of 0.20 nM; (5) some previously described VIP receptor antagonists including [4-Cl-D-Phe(6),Leu(17)]VIP, [Ac-Tyr(1),D-Phe(2)]GRF-(1-29) amide and VIP-(10-28) inhibited [I-125]VIP binding with a K-i of 0.7, 1.6 and 2.5 mu M, respectively, They failed to stimulate cAMP production in Clone 15 cells and inhibited, at least partially, the VIP (0.3 nM)-evoked cAMP production; (6) exposure of Clone 15 cells to 10 nM VIP for 24 h resulted in a sharp decrease in B-max in Clone 15 cells (0.43 vs. 1.62 pmol/mg protein in control cells) and in the potency and efficacy of VIP in stimulating cAMP. Moreover, immunofluorescence studies using confocal microscopy indicated that the receptor was internalized and sequestered in vesicular structure within the cells. It is concluded that Clone 15 cells provide a valuable tool to further characterize various functional and pharmacological aspects of the human VIP1 receptor.