DIFFERENTIAL ORIENTATION OF A GNRH AGONIST AND ANTAGONIST IN THE PITUITARY GNRH RECEPTOR

In the present study we have used a high affinity photoaffinity label (PAL) agonist 25-iodoTyr-D-Lys(para-N3-Benzoyl)-Leu-Arg-ProNHEt) and a PAL antagonist -D-Lys(para-N3-Benzoyl)Leu-Lys(Isp)-Pro-D-Ala-NH2) to covalently label the GnRH receptor. Rat pituitary membranes were incub ated 3 h with either the radioiodinated agonist or antagonist in the d ark, exposed to UV light, then electrophoresed in SDS. The PAL agonist and antagonist labeled broad bands (estimated molecular weight [M(r)] 46K-60K). Labeling by either PAL agonist or antagonist was displaced by unlabeled agonist or antagonist, indicating that the agonist and an tagonist bind to the same molecule. The broad band, believed to reflec t differential glycosylation, was divided into six sections correspond ing M(r) to 60K, 56K, 52K, 48K, 46K and 44K for the agonist and 62K, 5 8K, 54K, 52K, 48K and 45K for the antagonist; these were electroeluted with recovery > 80% based on radioactivity. Each could be re-electrop horesed to the same location from which it was eluted. Other eluted sa mples were treated with trypsin. The M(r) of the samples labeled with the agonist PAL were shifted to M(r) 48K, 42K, 40K, 37K, 35K and 33K b y proteolysis. The observation that each section shifted approximately the same M(r) after trypsin treatment suggests that the backbone of t he labeled proteins in each gel section is identical. Samples labeled with the antagonist PAL were shifted to M(r) < 10,000 in all cases. Th ese data indicate that the agonist and antagonist PALs bind to differe nt regions of the GnRH receptor and, therefore, are likely oriented di fferently with respect to die receptor and support the view that diffe rent strategies should be used for the design of agonists and antagoni sts.