LAL AF AATW ALY AFNA PFLN FOL ATI FPOC VILH YHLY FLAL ALW NYM EAVINGOAE COOH Figure 2 A transmembrane model of substance K receptor. Triangles, potential glycosylation sites; asterisks, possible phosphorylation sites; squared amino acids, hydrophobic amino acids in the putative transmembrane domains. the first indication that the neuropeptide receptor has sequence similarity with rhodopsin-type receptors and possesses multiple membrane-spanning domains. This structural characteristic also suggests that the substance K receptor is associated with a GTP-binding regulatory protein to effect the second messenger system involved in the substance K function. Thus, our results demonstrate that the structure and perhaps the function of the neuropeptide receptor are fundamentally similar to those of the receptors for classical small molecule transmitters such as epinephrine and acetylcholine. Future Directions Higher animals possess a large array of small neuropeptides, and many of these peptides are believed to play an important role in providing the specificity and complexity of interneuronal communications. Substance P is one of the best characterized neuropeptides and probably functions as a neuromediator involved in neurotransmission in primary sensory neurons. Although the physiological functions of substance K and neuromedin K are not well understood yet, they are present and produce effects in the nervous system and in peripheral tissues. Because the general feature of the preprotachykinin gene expressions has been elucidated, more detailed mechanisms for the regulation of the preprotachykinin genes could be investigated by using various molecular biological techniques such as DNA transfection and in vitro transcription and RNA splicing. Studies on the in vivo regulation of the preprotachykinin genes are also important for understanding the physiological roles of the tachykinin system. Because the conventional approach through protein purification had not readily been available for neuropeptide receptors, nothing was known about the molecular nature of the peptide receptors. The availability of the cloned cDNA for the substance K receptor would facilitate our understanding of some interesting aspects of neuropeptide receptors. These include the structure and function relationship of the receptor, the regulation of the receptor gene expression, intracellular signaling associated with the receptor function, and the ionic mechanisms underlying activation of the receptor response. Antibody against the receptor protein could also be made by chemically synthesizing a part of the amino acid sequence deduced from the cloned cDNA. The antibody thus formed will be used for determining the networks and physiological roles of peptidergic neurons. The oocyte expression system is capable of producing a number of foreign channels and their associated receptors, depending on the sources of mRNA injected (reviewed in Dascal 1987). The mRNA encoding a possible modulatory protein for a channel or a receptor may also be identified by the oocyte expression system combined with electrophysiological measurements. The molecular cloning method we developed will be widely applicable as a direct approach to characterize these proteins. REFERENCES Barnard, E.A.; Miledi, R.; and Sumikawa, K. Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proceedings of the Royal Society of London. Series B: Biological Sciences (London) 215(1199):241-246, 1982. Dascal, N. The use of Xenopus oocytes for the study of ion channels. CRC Critical Reviews in Biochemistry 22(4):317-387, 1987. Dohlman, H.G.; Caron, M.G.; and Lefkowitz, R.J. A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry 26(10):2657-2664, 1987. Harada, Y.; Takahashi, T.; Kuno, M.; Nakayama, K.; Masu, Y.; and Nakanishi, S. Expression of two different tachykinin receptors in Xenopus oocytes by exogenous mRNAs. Journal of Neuroscience 7(10):3265-3273, 1987. Kawaguchi, Y.; Hoshimaru, M.; Nawa, H.; and Nakanishi, S. Sequence analysis of cloned cDNA for rat substance P precursor: Existence of a third substance P precursor. Biochemical and Biophysical Research Communications 139(3):10401046, 1986. Kotani, H.; Hoshimaru, M.; Nawa, H.; and Nakanishi, S. Structure and gene organization of bovine neuromedin K precursor. Proceedings of the National Academy of Sciences of the United States of America 83(18):7074-7078, 1986. Masu, Y.; Nakayama, K.; Tamaki, H.; Harada, Y.; Kuno, M.; and Nakanishi, S. cDNA cloning of bovine substance-K receptor through oocyte expression system. Nature 329(6142):836-838, 1987. Nakanishi, S. Structure and regulation of the preprotachykinin gene. Trends in Neurosciences 9:41-44, Jan. 1986. Nakanishi, S. Substance P precursor and kininogen: Their structures, gene organizations, and regulation. Physiological Reviews 67(4):1117-1142, 1987. Nawa, H.; Doteuchi, M.; Igano, K.; Inouye, K.; and Nakanishi, S. Substance K: A novel mammalian tachykinin that differs from substance P in its pharmacological profile. Life Sciences 34(12):1153-1160, 1984. Nawa, H.; Hirose, T.; Takashima, H.; Inayama, S.; and Nakanishi, S. Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor. Nature 306(5938):32-36, 1983. Nawa, H.; Kotani, H.; and Nakanishi, S. Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 312(5996): 729-734, 1984. Quirion, R. Multiple tachykinin receptors. Trends in Neurosciences 8:183-185, May 1985. Molecular Mechanisms Dictating Cell-Specific Patterns of Neuroendocrine Gene Expression R.B. Emeson*, J.W. Guise+, E.B. Crenshaw III*, A.F. Russo, and M.G. Rosenfeld +# + Eukaryotic Regulatory Biology Program, School of Medicine San Diego, CA 92093 Howard Hughes Medical Institute Our goal has been to understand the tissue-specific expression of eukaryotic transcription units encoding neuroendocrine genes because many of these genes are expressed in both the endocrine and central nervous systems. Based upon analyses of the rat and human calcitonin/calcitonin gene-related peptide (CGRP) genes, alternative RNA processing has been demonstrated to represent an important developmental strategy by which the neuroendocrine system may dictate a tissue-specific pattern of peptide hormone production. We have initiated an analysis of the molecular mechanisms responsible for generating such restricted patterns of posttranscriptionally regulated gene expression to provide general insights into the molecular strategies critical for development and function of the neuroendocrine system. The calcitonin/CGRP gene represents a complex transcriptional unit in which cell-specific alternative RNA processing results in the production of calcitonin and CGRP messenger RNA (mRNA) in thyroid C cells and neurons, respectively. The rat calcitonin/CGRP gene comprises six exons; calcitonin mRNA is produced by splicing the first three exons to the fourth exon, representing >98 percent of the mature transcripts from this gene in thyroid C cells. In the brain and peripheral nervous system, however, the first three exons are spliced to the fifth and sixth exons, generating an mRNA species encoding the precursor to a novel 37-amino acid neuropeptide, referred to as calcitonin gene-related peptide. CGRP mRNA represents >95 percent of the mature transcripts generated from this gene in the central and peripheral nervous systems. The distribution of CGRP-containing cells and pathways in the brain and other tissues, accompanied by a number of physiological studies, has suggested functions for this peptide in nociception, ingestive behavior, and modulation of the cardiovascular and endocrine systems. A Single Neuroendocrine Gene Generates Multiple RNA Products Via Alternative RNA Processing Events Molecular cloning of DNA complementary to rat calcitonin mRNA predicted the structure of the polypeptide precursor to the 32-amino acid calcium-regulating hormone, calcitonin (Rosenfeld et al. 1981; Amara et al. 1982). A second, structurally distinct transcript, referred to as calcitonin gene-related peptide mRNA (CGRP mRNA) (Rosenfeld et al. 1982; Amara et al. 1982; Rosenfeld et al. 1983) was first noted during the spontaneous "switching" of serially transplanted rat medullary thyroid carcinomas (MTCs) from states of "high" to "low" calcitonin production. Isolation and sequence analysis of the calcitonin genomic DNA as well as calcitonin and CGRP cDNAs demonstrated that both CGRP and calcitonin transcripts were generated by differential RNA processing from a single genomic locus (Amara et al. 1982; Rosenfeld et al. 1983). CGRP and calcitonin mRNAs share sequence identity through nucleotide 227 of the coding region, predicting that the initial 72 N-terminal amino acids of each precursor are identical, but then diverge entirely in nucleotide sequence, encoding unique C-terminal domains (Amara et al. 1982). Posttranslational protein processing signals within the C-terminal region of CGRP predict the production of a 37-amino acid polypeptide containing a C-terminal amidated phenylalanine residue (Amara et al. 1982). Based upon the structure of the calcitonin/CGRP gene, production of calcitonin mRNA involves splicing of the first three exons, present in both mRNAs, to the fourth exon, which encodes the entire calcitonin coding region as well as 3' noncoding sequences. Alternative splicing of the first three exons to the fifth and sixth exons, which contain the entire CGRP coding sequence and 3' noncoding sequences, respectively, results in production of CGRP mRNA; in this case, the fourth exon is excised along with the flanking intervening sequences. Calcitonin Gene-Related Peptide Is the Product of Using antisera generated against a synthetic peptide corresponding to the fourteen C-terminal amino acids of CGRP, immunoreactive CGRP demonstrated a unique anatomical distribution in the central nervous system, |