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 (pGlu-His-Trp-Ser-125I-iodoTyr-D-Lys(para-N3-Benzoyl)-Leu-Arg-P roNHEt) and a PAL antagonist (NAcD2Nal-D4ClPhe-D3Pal-Ser-125I-iodo-NMeTyr-D-L ys(para-N3-Benzoyl)Leu-Lys(Isp) - Pro-D-Ala-NH2) to covalently label the GnRH receptor. Rat pituitary membranes were incubated 3 h with either the radioiodinated agonist or antagonist in the dark, 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 antagonist bind to the same molecule. The broad band, believed to reflect differential glycosylation, was divided into six sections corresponding M(r) to 60K, 56K, 52K, 48K, 46K and 44K for the agonist and 62K, 58K, 54K, 52K, 48K and 45K for the antagonist; these were electroeluted with recovery > 80% based on radioactivity. Each could be re-electrophoresed to the same location from which it was eluted. Other eluted samples 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 by proteolysis. The observation that each section shifted approximately the same M(r) after trypsin treatment suggests that the backbone of the labeled proteins in each gel section is identical. Samples labeled with the antagonist PAL were shifted to M(r) < 10,000 in all cases. These data indicate that the agonist and antagonist PALs bind to different regions of the GnRH receptor and, therefore, are likely oriented differently with respect to the receptor and support the view that different strategies should be used for the design of agonists and antagonists.