THE DISULFIDE-BONDED LOOP OF CHROMOGRANINS, WHICH IS ESSENTIAL FOR SORTING TO SECRETORY GRANULES, MEDIATES HOMODIMERIZATION

Chromogranins A and B, two widespread neuroendocrine secretory protein s, contain a homologous N-terminal disulfide-bonded loop that is requi red for sorting to secretory granules. Here we have investigated the r ole of this loop in the oligomerization of chromogranin A. Reduction o f the disulfide bond or the addition of an excess of an N-terminal chr omogranin A fragment containing the loop (CgA(1-60)) resulted in the d issociation into monomers of the chromogranin A dimer found at pH 7.4 and 6.4 and of the chromogranin tetramer found at pH 5.4. The addition of an excess of a synthetic peptide corresponding to the conserved C- terminal domain of chromogranin A (CgA(406-431)) had no effect on the chromogranin dimers at pH 7.4 and 6.4 and resulted in the dissociation of the chromogranin A tetramers at pH 5.4 into dimers. Fluorescence e nergy transfer experiments using fluorescently labeled CgA(1-60) showe d that the N-terminal disulfide-bonded loop has a high affinity for ho modimerization (K-D = 20 nM at pH 6.4), which was sufficient to mediat e dimerization of full-length chromogranin A. Association and dissocia tion of loop-mediated chromogranin A dimerization approached completio n within a few seconds. Our results imply that chromogranin A homodime rizes shortly after synthesis in the endoplasmic reticulum and that th e loop-mediated homodimeric state is an essential prerequisite for its sorting, in the trans-Golgi network to secretory granules.