Discovery of a new molecular form of neuropeptide Y from procine brain, NPY 3–36, which selectively binds to Y2 receptors

161 DISCOVERY OF A NEW MOLECULAR FORM OF NEUROPEPTIDE Y FROM PROCINE BRAIN, NPY 3-36, WHICH SELECTIVELY BINDS TO Y2 RECEPTORS Grandt D., SchimiczekM., Feth F., AhrensO., RascherW., LayerP., Goebell H., Davis M, ShivelyJE., ReeveJR. Jr., EysseleinVE. Univ. of Essen,Germany;HarborUCLA,CUREVA Wadsworthand City of HopeRes.lnst. Duarte,USA Neuropeptide (NPY 1-36) is a neurotransmitter highly abundant in the CNS as well as in peripheral neurons of the gut. Y1 and Y2 receptors have been described to which NPY and Peptide YY (PYY 1-36) bind with similar affinity. The physiological importance of receptor heterogenity is unknown, as the endogenous agonists, NPY/PYY 1-36, binds equally to both receptor subtypes. We have previously characterized a second molecular form of PYY, PYY 3-36 in human and canine intestine, which selectively binds to Y2 receptors. We now report the purification of a new molecular form of NPY from porcine brain and characterize its binding to Y1 and Y2 receptors. Methods: (1) NPY was purified from porcine brain by sequential steps of reverse-phase HPLC and ion-exchange FPLC. Purified NPY was characterized by microsequence and mass spectral analysis. (2) Competitive receptor binding of synthetic NPY and NPY 3-36 to Y1 receptors on SK-N-MC cells and Y2 receptors on porcine splenic membrane preparations was studied. Results: NPY-immunoreactivity eluted in two peaks on a phenyl r.p. HPLC column. Microsequence analysis and mass spectral analysis were consistent with NPY 3-36 and NPY 1-36. Receptor binding studies demonstrate that NPY 3-36 selectively binds to Y2 receptors on splenic membrane preparations with almost the same affinity as NPY 1-36. NPY 3-36 was about 1000 fold less potent on Y1 receptors on SK-N-C cells than NPY 1-36. Conclusions: NPY 3-36 is abundant in porcine brain. It selectively binds to Y2 receptors. The existence of a Y2-selective agonist suggests a physiological importance of Y receptor heterogenity for NPY.

PEPTIDE YY 3-36 IS AN ENDOGENOUS Y2 SELECTIVE AGONIST AND A MAJOR MOLECULAR FORM OF PYY IN CIRCULATING BLOOD IN MAN Grandt D. Schimiczek M., Feth F., Ahrens O. RascherW., Goebell H., ReeveJr Jr., Michel MC, Eysselein VE Universityof Essen, Germany, Harbor UCLAand CURE VA Wadsworth, USA The importance of receptor heterogenity for Peptide YY is unclear, as the endogenous agonist, PYY 1-36, binds to both receptor subtypes (Y1,Y2) with similar affinity. We have previously purified a second molecular form of PYY, PYY 3-36, from human intestine. We now report the abundance of PYY 3-36 in circulating blood and characterize its affinity to Y1 and Y2 receptors. Methods: PYY from fastened and postprandial human Fig.1 plasma was studied using HPLC and RIA. Synthetic Z li a ' I. . human PYY 1-36 and PYY 3-36 were used as standards. Binding of PYY 3-36 to Y1 receptors was studied in SKN-MC cells. Procine splenic membrane preparations 4O were used for studying binding to Y2 receptors. Results: PYY-IR in human plasma eluted in two peaks on a r.p. phenyl HPLC column. Coelution identifies them I0"*=10-ii10-i0%0-9 lO*ll 10-7 I0 "a' as PYY 1-36 and PYY 3-36. PYY 3-36 accounts for 37% [I,,,J,t hi1 |. H _+ 7.8 of PYY-IR in the fastened state and 54 % _ 5.3 in the postprandial state (n=4). Receptor binding studies Fig. 2 showed that PYY 3-36 has a similar affinity as PYY 1-36 to Y2 receptors on splenic membrane preparations (Fig. 1), but is less than 1000 fold potent on Y | receptors of 4010 ?yy l-M, SK-N-MC cells (Fig. 2). , 20{0 ?YY '-3¢ ~,~ Conclusions: The postprandial increase in plasma PYYimmunoreactivity is predominantly due to an increase in O[ ~<-H-HC PYY 3-36, which selectively binds to Y2 receptors. IO-':~)O-,qO-tOlO-9 tO-O i0-7 ZO-6 ll,,,i,t I,h, ]. IS Proteolytic processing of PYY 1-36 to form PYY 3-36 generates receptor selectivity for PYY.