Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH 2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the ␣-melanocyte stimulating hormone analogue [Nle 4 ,D-Phe 7 ] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.

The turnover of cells in renewing epithelia presents an opportunity to examine cell death pathways in adult vertebrates. In mouse lingual epithelium a typical taste receptor cell survives for 9 days, until it is killed by an unknown cascade of death factors. Apoptosis was implicated by the presence of fragmented DNA in about 8% of taste receptor cells in the vallate papilla. In using immunocytochemistry to seek putative death factors, we observed that squamous epithelial cells of the tongue were negative for Bax, a death factor in the Bcl-2 family of survival/death factors, and were also negative for p53, a tumor-suppressor protein linked to apoptosis and Bax transcription. In contrast, 8-10% of the taste receptor cells were Bax-positive, and 9-11% were p53 positive. These immunopositive taste receptor cells were more likely to display death-related morphologic defects than other receptor cells, and they frequently coexpressed p53 and Bax. In both neonatal and adult mice, the labeling of dividing cells with 5-bromo-2Ј-deoxyuridine indicated that all Bax-positive taste cells were at least 5 days old. On postnatal day 7, when few taste cells were old, no more than 1% of taste cells were immunopositive for either p53 or Bax. We inferred that old taste receptor cells employ p53 and Bax as part of their apoptotic death pathway. The routine expression of p53 by postmitotic, aged taste cells broadens the conventional view that p53 is restricted to mitotic cells that have stress-damaged DNA. Furthermore, the scattered distribution of aged receptor cells within the taste bud excludes some explanations for stable taste signals during receptor cell turnover.

All or nearly all intragemmal (elongated) cells of rat taste buds were immunopositive for keratins 7, 8, and 19. In contrast, keratin 18 was detected in 19 +/- 5 cells per taste bud (mean +/- sd), or about 25% of the intragemmal cells. During taste bud development keratins 7, 8, and 19 were evident initially in polygonal cells and later in elongated taste cells. Keratin 8 appeared in vallate taste cells at P0 (postnatal day 0), followed by keratins 7 and 19 at P1, and keratin 18 at P2-P3. Keratin 18 was always limited to elongated cells. The assemblage of elongated taste cells comprising a taste bud began with a single elongated cell, rather than with the synchronous elongation of a cluster of cells. Developmental errors were observed at P2-P3, e.g., some vallate taste cells had a misoriented axis. In order to study the pace of keratin differentiation during cell turnover we injected bromodeoxyuridine (BrdU) into adult rats to monitor taste cell age. Keratin-19-positive intragemmal cells differentiated within 1 day. In contrast, keratin 18 was first detected in cells aged 3 days. Hence, both in taste cell development and replacement, keratin 18 was restricted to the older cells; it was the last taste cell keratin to become expressed during differentiation.

In the adult mouse tongue, an average of 11% of the gustatory receptor cells are replaced each day. In investigating homeostatic cell death mechanisms in gustatory renewing epithelium, we observed that taste receptor cells were selectively immunopositive for the bcl-2 family death factor, Bax, and for the protease Caspase-2 (Nedd2/Ich1). We determined that 8-10% of the taste receptor cells of the vallate papilla were Bax positive and that 11% were Caspase-2 positive. Some of these immunopositive taste cells had apoptotic morphological defects. Within the subset of vallate taste cells immunopositive for either Caspase-2 or Bax, up to 79% coexpressed both death factors. Bax and Caspase-2 first appeared in occasional vallate taste receptor cells on the same postnatal day-the day after birth. bax null mutation markedly reduced gustatory Caspase-2 immunoexpression. These observations suggest that taste cell death pathways utilize p53, Bax, and Caspase-2 to dispose of aged receptor cells. Apart from reducing Caspase-2 expression, Bax deficiency also altered taste organ development. bax(-/-) mice had a more profusely innervated vallate papilla, which grew to be 25% longer and taller, with the mean taste bud containing more than twice the normal number of taste cells. This augmentation of taste organ development with increased innervation is complementary to the well-documented reduction in taste organ development with sparse innervation. We propose that additional taste neurons survived programmed cell death in Bax-deficient mice, thereby providing an inductive boost to vallate gustatory development.

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