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 recom binant human vasoactive intestinal peptide (VIP), receptor. A clone re ferred 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 membran es 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 pepti des was: VIP (0.9 nM) = pituitary adenylate cyclase-activating peptide (PACAP)-27 (1.3 nM) < PACAP-38 (6.8 nM) < helodermin (46.0 nM) < huma n 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 recombina nt receptor; (4) VIP stimulated cAMP production in Clone 15 cells an E C(50) of 0.20 nM; (5) some previously described VIP receptor antagonis ts 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 produc tion 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 V IP 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 stud ies using confocal microscopy indicated that the receptor was internal ized and sequestered in vesicular structure within the cells. It is co ncluded that Clone 15 cells provide a valuable tool to further charact erize various functional and pharmacological aspects of the human VIP1 receptor.