Abstract
The neuroendocrine granule-associated protein 7B2, unlike many other neuroendocrine precursor proteins stored in secretory granules, carries in its primary structure the Arg-Xaa-Arg/Lys-Arg processing site usually found in constitutively secreted precursor proteins and recognized by the ubiquitously expressed convertase, furin. pro7B2 (30 kDa), when expressed in endocrine (AtT-20, PC12, and GH4C1) or non-endocrine (Ltk-) cell lines using recombinant vaccinia viruses, was converted to a 23-kDa form. Mutation of the P4 Arg to Gly completely prevented this conversion. When excess pro7B2 was coexpressed with the pro-protein convertases PC1, PC2, or furin, only furin could induce complete processing. In addition, coexpression of pro7B2 in LoVo cells, which are devoid of endogenous furin activity, with each one of the three convertases, showed that only furin was able to induce processing of this precursor. pro7B2 processing in AtT-20 was completely abolished when protein transport into Golgi compartments was blocked by cell incubation at either 15 or 37 degrees C in the presence of monensin or brefeldin A. Furthermore, pulse-chase experiments in the presence of Na2[35S]SO4 showed that pro7B2 is Tyr-sulfated in the trans-Golgi network before it is processed. These results demonstrate that pro7B2 is first processed by a furin-like enzyme within the trans-Golgi network into a 23-kDa form that is then sequestered into secretory granules.
Highlights
From the Laboratories of $Structure and Metabolism of Neuropeptides, $Molecular Neuroendocrinology,and IWeuroendocrine Biochemist“r,v. theClinical Research Institute of Montre‘al, Universityof Montreal, Montre‘al, Quebec H2W lR7, Canada
The neuroendocrine granule-associatedprotein 7B2, terization, and sequencing, we have previously shown that in unlike many other neuroendocrine precursor proteins AtT-20 cells, pro7B2 processing occurs at this particular site stored in secretory granules, carries inits primary yielding a shorter 23-kDa form(11).thecomplete structure the Arg-Xaa-ArgILys-Argprocessing site usu- sequencing of a150-residue 7B2 protein (23 ma) extracted ally found in constitutively secreted precursor proteins from porcine pituitary (12) supported thisconclusion
Because of the high degree of conservation of its sequence during evolution (90% sequencesimilarity betweenxenopuslaevis and man(5)), an importantphysiological role for this molecule has been postulated but not yet elucidat7eBd.2 produced in neuroendocrine cells is stored in secretory granules, and its secretion can be induced by secretagogues (6, 7)
Summary
Were first treated with a polyclonal anti-serum directed against the COOH-terminal region of either convertases. Analysis of the pulse-labeled material secreted served in thecases ofAtT-20, GH,Cl, and PC12 cells, indicating into the medium demonstrated that LoVo cells are unable to that there was still enough 7B2-processing activity in these process pro7B2 unless they araelso supplemented withrecomcells even after overnight incubations (datanot shown).Under binant furin(Fig. 5), in which case all the pro7B2 is converted similar conditions, Ltk- cells secreted relatively high amounts into the23-kDa form. It should be notedthat the23-kDa form of pro7B2 in themedium.
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