A receptor in pituitary and hypothalamus that functions in growth hormone release

Science

Volumn 273, Issue 5277, 1996, Pages 974-977

  Howard, Andrew D ^a   Feighner, Scott D ^a   Cully, Doris F ^a   Arena, Joseph P ^a   Liberator, Paul A ^a   Rosenblum, Charles I ^a   Hamelin, Michel ^a   Hreniuk, Donna L ^a   Palyha, Oksana C ^a   Anderson, Jennifer ^a   Paress, Philip S ^a   Diaz, Carmen ^a   Chou, Michael ^a   Liu, Ken K ^a   McKee, Karen Kulju ^a   Pong, Sheng Shung ^a   Chaung, Lee Yuh ^a   Elbrecht, Alex ^a   Dashkevicz, Mike ^a   Heavens, Robert ^b   Rigby, Mike ^b   Sirinathsinghji, Dalip J S ^b   Dean, Dennis C ^a   Melillo, David G ^a   Patchett, Arthur A ^a   Nargund, Ravi ^a   Griffin, Patrick R ^a   DeMartino, Julie A ^a   Gupta, Sunil K ^a   Schaeffer, James M ^a   Smith, Roy G ^a   Van Der Ploeg, Lex H T ^a   more..

^a MERCK RESEARCH LABORATORIES   (United States)

^b NEUROSCIENCE RESEARCH CENTRE   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

GUANINE NUCLEOTIDE BINDING PROTEIN; HORMONE RECEPTOR;

ARTICLE; GROWTH HORMONE RELEASE; HORMONAL REGULATION; HORMONE ACTION; HYPOPHYSIS; HYPOTHALAMUS; MOLECULAR CLONING; PRIORITY JOURNAL; SWINE;

SUS SCROFA;

EID: 9444246501     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5277.974     Document Type: Article

References (35)

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18. 6 independent clones; 95% inserts; insert size ∼1.65 kb; M. P. Kriegler, Gene Transfer and Expression: A Laboratory Manual (Stockton Press, New York, 1990)]. We synthesized cRNA from Not I-digested DNA by using, with modifications, a kit from Promega Biotech [J. P. Arena et al., Mol. Pharmacol. 40, 368 (1991)].
19. 6 independent clones; 95% inserts; insert size ∼1.65 kb; M. P. Kriegler, Gene Transfer and Expression: A Laboratory Manual (Stockton Press, New York, 1990)]. We synthesized cRNA from Not I-digested DNA by using, with modifications, a kit from Promega Biotech [J. P. Arena et al., Mol. Pharmacol. 40, 368 (1991)].
20. 6 independent clones; 95% inserts; insert size ∼1.65 kb; M. P. Kriegler, Gene Transfer and Expression: A Laboratory Manual (Stockton Press, New York, 1990)]. We synthesized cRNA from Not I-digested DNA by using, with modifications, a kit from Promega Biotech [J. P. Arena et al., Mol. Pharmacol. 40, 368 (1991)].
21. 6 independent clones; 95% inserts; insert size ∼1.65 kb; M. P. Kriegler, Gene Transfer and Expression: A Laboratory Manual (Stockton Press, New York, 1990)]. We synthesized cRNA from Not I-digested DNA by using, with modifications, a kit from Promega Biotech [J. P. Arena et al., Mol. Pharmacol. 40, 368 (1991)].
22. 2+ -free OR-2 medium. Each cRNA pool was tested in triplicate. Measurements (duration 2 min) were triggered by the injection of 0.1 ml of 30 μM MK-0677.
23. 2+ -free OR-2 medium. Each cRNA pool was tested in triplicate. Measurements (duration 2 min) were triggered by the injection of 0.1 ml of 30 μM MK-0677.
24. 2+ -free OR-2 medium. Each cRNA pool was tested in triplicate. Measurements (duration 2 min) were triggered by the injection of 0.1 ml of 30 μM MK-0677.
25. 2+ -free OR-2 medium. Each cRNA pool was tested in triplicate. Measurements (duration 2 min) were triggered by the injection of 0.1 ml of 30 μM MK-0677.
26. 2+ -free OR-2 medium. Each cRNA pool was tested in triplicate. Measurements (duration 2 min) were triggered by the injection of 0.1 ml of 30 μM MK-0677.
27. 6 phages gave 21 GHS-R clones]. DNA sequencing was done on both strands [automated Applied Biosystems instrument (ABI model 373); manually by dideoxy chain termination (Sequenase version 2.0; U.S. Biochemical, Cleveland, OH)]. Database searches [GenBank 92, EMBL 43, Swiss-Prot 31, PIR 45, dEST (Gbest 92), and Prosite 12], sequence alignments, and analysis of the GHS-R nucleotide and protein sequences were done with the GCG Sequence Analysis Software (Madison, Wl; pileup, peptide structure and motif programs), FASTA and BLAST search programs, the PC/Gene software suite from Intelligenetics (San Francisco, CA; protein analysis programs), and Lasergene software (DMA Star, Madison, Wl).
28. 35S]MK-0677 (0.05 to 1 nM), 10 μl of competing drug, and 380 to 390 μl of homogenization buffer. Specific binding (>90% of total) equaled the difference between total and nonspecific binding obtained in the presence of 50 nM unlabeled MK-0677.
29. 9 cpm/μg), were each 45 bases long and antisense to nucleotides 855 through 909 and 979 through 1023. Hybridizations of rhesus brain sections were done as described [D. J. S. Sirinathsinghji et al., Neuroscience 34, 675 (1990); D. J. S. Sirinathsinghji and S. B. Dunnet, in Molecular Imaging in Neuroscience, N. Sharif, Ed. (Oxford Univ. Press, Oxford. 1993), p. 43]. After hybridization, the sections were washed for 1 hour in 1 × SSC at 57°C, briefly rinsed in 0.1 × SSC and dehydrated in 70% and 95% ethanol, air-dried, and then exposed to Hyperfilm β-max x-ray film (Amersham) for 7 days. Adjacent slide-mounted sections incubated with labeled oligonucleotide probe in the presence of a 100-fold excess of unlabeled oligonucleotide probe or with a sense probe from the same region produced no hybridization signal.
30. 9 cpm/μg), were each 45 bases long and antisense to nucleotides 855 through 909 and 979 through 1023. Hybridizations of rhesus brain sections were done as described [D. J. S. Sirinathsinghji et al., Neuroscience 34, 675 (1990); D. J. S. Sirinathsinghji and S. B. Dunnet, in Molecular Imaging in Neuroscience, N. Sharif, Ed. (Oxford Univ. Press, Oxford. 1993), p. 43]. After hybridization, the sections were washed for 1 hour in 1 × SSC at 57°C, briefly rinsed in 0.1 × SSC and dehydrated in 70% and 95% ethanol, air-dried, and then exposed to Hyperfilm β-max x-ray film (Amersham) for 7 days. Adjacent slide-mounted sections incubated with labeled oligonucleotide probe in the presence of a 100-fold excess of unlabeled oligonucleotide probe or with a sense probe from the same region produced no hybridization signal.
31. D. J. S. Sirinathsinghji, R. Heavens, M. Rigby, L. H. T. Van der Ploeg, unpublished data.
32. D. J. S. Sirinathsinghji et al., Neuroreport 6, 1989 (1995).
33. S. L. Dickson, O. Doutrelant-Viltart, G. Leng, J. Endocrinol. 146, 519 (1995).
34. Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr: V, Val; W, Trp; and Y, Tyr.
35. We thank B. DeFranco, K. Judd, E. Szekely, and D. Thompson (Branchburg Farm) for swine tissues; D. Boltz and A. Sirotina for discussion and fluorescence-activated cell sorting analysis; D. Button and M. Brownstein (NIH) for aequorin cDNAs; C. Y. Bowers (Tulane Medical School) for GHRP-2; K. Elliston for sequence analysis; D. Leong for helpful discussion; K. Prendergast and D. Underwood for helpful discussion and modeling of the GHS binding site; Y. Karkhanis for antibody analysis; J. Ngai, T. Livelli, and colleagues (Molecular Cell Sciences) for support in the production of cDNA libraries and advice; K. Likowski for expert secretarial assistance; and MRL Visual Communications for preparation of figures. The care of animals was in accordance with institutional guidelines.