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Juxtamembrane Region of the Amino Terminus of the Corticotropin Releasing Factor Receptor Type 1 Is Important for Ligand Interaction
The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the N...
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Published in: | Biochemistry (Easton) 2001-02, Vol.40 (5), p.1187-1195 |
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description | The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3−34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1−34). CRF and PTH(1−34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1−31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC−F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys68-Glu109 domain is important for binding and that the Cys87-Cys102 region plays an important role in CRFR1 activation. |
doi_str_mv | 10.1021/bi001758y |
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The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3−34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1−34). CRF and PTH(1−34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1−31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC−F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys68-Glu109 domain is important for binding and that the Cys87-Cys102 region plays an important role in CRFR1 activation.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi001758y</identifier><identifier>PMID: 11170443</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amino Acid Sequence ; Amphibian Proteins ; Animals ; COS Cells ; Cyclic AMP - metabolism ; Ligands ; Membrane Proteins - chemistry ; Membrane Proteins - genetics ; Membrane Proteins - metabolism ; Membrane Proteins - physiology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Mutagenesis, Insertional ; Peptide Fragments - chemistry ; Peptide Fragments - genetics ; Peptide Fragments - metabolism ; Peptide Fragments - physiology ; Peptide Hormones ; Peptides - pharmacology ; Protein Structure, Tertiary - genetics ; Proto-Oncogene Proteins c-myc - genetics ; Rats ; Receptor, Parathyroid Hormone, Type 1 ; Receptors, Corticotropin-Releasing Hormone - chemistry ; Receptors, Corticotropin-Releasing Hormone - genetics ; Receptors, Corticotropin-Releasing Hormone - metabolism ; Receptors, Corticotropin-Releasing Hormone - physiology ; Receptors, Parathyroid Hormone - chemistry ; Receptors, Parathyroid Hormone - genetics ; Receptors, Parathyroid Hormone - metabolism ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - metabolism ; Structure-Activity Relationship</subject><ispartof>Biochemistry (Easton), 2001-02, Vol.40 (5), p.1187-1195</ispartof><rights>Copyright © 2001 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a349t-33f9a9fa6af53d812f64a6e733fd82e505de4a1fc1eb3267cd555915c3f3dabd3</citedby><cites>FETCH-LOGICAL-a349t-33f9a9fa6af53d812f64a6e733fd82e505de4a1fc1eb3267cd555915c3f3dabd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11170443$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Assil, Iman Q</creatorcontrib><creatorcontrib>Qi, Lai Jun</creatorcontrib><creatorcontrib>Arai, Maya</creatorcontrib><creatorcontrib>Shomali, Mansur</creatorcontrib><creatorcontrib>Abou-Samra, Abdul B</creatorcontrib><title>Juxtamembrane Region of the Amino Terminus of the Corticotropin Releasing Factor Receptor Type 1 Is Important for Ligand Interaction</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3−34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1−34). CRF and PTH(1−34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1−31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC−F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys68-Glu109 domain is important for binding and that the Cys87-Cys102 region plays an important role in CRFR1 activation.</description><subject>Amino Acid Sequence</subject><subject>Amphibian Proteins</subject><subject>Animals</subject><subject>COS Cells</subject><subject>Cyclic AMP - metabolism</subject><subject>Ligands</subject><subject>Membrane Proteins - chemistry</subject><subject>Membrane Proteins - genetics</subject><subject>Membrane Proteins - metabolism</subject><subject>Membrane Proteins - physiology</subject><subject>Mice</subject><subject>Mice, Inbred BALB C</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Insertional</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - metabolism</subject><subject>Peptide Fragments - physiology</subject><subject>Peptide Hormones</subject><subject>Peptides - pharmacology</subject><subject>Protein Structure, Tertiary - genetics</subject><subject>Proto-Oncogene Proteins c-myc - genetics</subject><subject>Rats</subject><subject>Receptor, Parathyroid Hormone, Type 1</subject><subject>Receptors, Corticotropin-Releasing Hormone - chemistry</subject><subject>Receptors, Corticotropin-Releasing Hormone - genetics</subject><subject>Receptors, Corticotropin-Releasing Hormone - metabolism</subject><subject>Receptors, Corticotropin-Releasing Hormone - physiology</subject><subject>Receptors, Parathyroid Hormone - chemistry</subject><subject>Receptors, Parathyroid Hormone - genetics</subject><subject>Receptors, Parathyroid Hormone - metabolism</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Structure-Activity Relationship</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><recordid>eNptkD1PwzAQhi0EgvIx8AeQFwaGgB3HSTOiikJRJRCU2bok52IgdmS7Et354RiVj4Xp7Pee9073EnLM2TlnOb9oDGO8kuP1FhlxmbOsqGu5TUaMsTLL65Ltkf0QXtK3YFWxS_Y45xUrCjEiH7er9wg99o0Hi_QBl8ZZ6jSNz0gve2MdXaBPdRV-1Inz0bQuejcYmxxvCMHYJZ1CG51PQovD12OxHpByOgt01g_JAzZSnfS5WYLt6MxG9MmS9h2SHQ1vAY--6wF5ml4tJjfZ_O56NrmcZyCKOmZC6BpqDSVoKboxz3VZQIlV0rtxjpLJDgvguuXYiLys2k5KWXPZCi06aDpxQM42c1vvQvCo1eBND36tOFNfSarfJBN7smGHVdNj90d-R5eAbAOYEPH9tw_-VZWVqKRa3D-qa1lPy-lEKJn40w0PbVAvbuVtOvWfxZ97NItv</recordid><startdate>20010206</startdate><enddate>20010206</enddate><creator>Assil, Iman Q</creator><creator>Qi, Lai Jun</creator><creator>Arai, Maya</creator><creator>Shomali, Mansur</creator><creator>Abou-Samra, Abdul B</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>20010206</creationdate><title>Juxtamembrane Region of the Amino Terminus of the Corticotropin Releasing Factor Receptor Type 1 Is Important for Ligand Interaction</title><author>Assil, Iman Q ; Qi, Lai Jun ; Arai, Maya ; Shomali, Mansur ; Abou-Samra, Abdul B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a349t-33f9a9fa6af53d812f64a6e733fd82e505de4a1fc1eb3267cd555915c3f3dabd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Amino Acid Sequence</topic><topic>Amphibian Proteins</topic><topic>Animals</topic><topic>COS Cells</topic><topic>Cyclic AMP - metabolism</topic><topic>Ligands</topic><topic>Membrane Proteins - chemistry</topic><topic>Membrane Proteins - genetics</topic><topic>Membrane Proteins - metabolism</topic><topic>Membrane Proteins - physiology</topic><topic>Mice</topic><topic>Mice, Inbred BALB C</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Insertional</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - metabolism</topic><topic>Peptide Fragments - physiology</topic><topic>Peptide Hormones</topic><topic>Peptides - pharmacology</topic><topic>Protein Structure, Tertiary - genetics</topic><topic>Proto-Oncogene Proteins c-myc - genetics</topic><topic>Rats</topic><topic>Receptor, Parathyroid Hormone, Type 1</topic><topic>Receptors, Corticotropin-Releasing Hormone - chemistry</topic><topic>Receptors, Corticotropin-Releasing Hormone - genetics</topic><topic>Receptors, Corticotropin-Releasing Hormone - metabolism</topic><topic>Receptors, Corticotropin-Releasing Hormone - physiology</topic><topic>Receptors, Parathyroid Hormone - chemistry</topic><topic>Receptors, Parathyroid Hormone - genetics</topic><topic>Receptors, Parathyroid Hormone - metabolism</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Structure-Activity Relationship</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Assil, Iman Q</creatorcontrib><creatorcontrib>Qi, Lai Jun</creatorcontrib><creatorcontrib>Arai, Maya</creatorcontrib><creatorcontrib>Shomali, Mansur</creatorcontrib><creatorcontrib>Abou-Samra, Abdul B</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Assil, Iman Q</au><au>Qi, Lai Jun</au><au>Arai, Maya</au><au>Shomali, Mansur</au><au>Abou-Samra, Abdul B</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Juxtamembrane Region of the Amino Terminus of the Corticotropin Releasing Factor Receptor Type 1 Is Important for Ligand Interaction</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2001-02-06</date><risdate>2001</risdate><volume>40</volume><issue>5</issue><spage>1187</spage><epage>1195</epage><pages>1187-1195</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>The functional properties of the amino terminus (NT) of the corticotropin releasing factor (CRF) receptor type 1 (R1) were studied by use of murine (m) CRFR1 and rat (r) parathyroid hormone (PTH)/parathyroid hormone-related peptide receptor (PTH1R) chimeras. The chimeric receptor CXP, in which the NT of mCRFR1 was annealed to the TMs of PTH1R, and the reciprocal hybrid, PXC, bound radiolabeled analogues of sauvagine and PTH(3−34), respectively. Neither hybrid bound radiolabeled CRF or PTH(1−34). CRF and PTH(1−34) weakly stimulated intracellular cAMP accumulation in COS-7 cells transfected with PXC and CXP, respectively. Thus the NT is required for ligand binding and the TMs are required for agonist-stimulated cAMP accumulation. Replacing individual intercysteine segments of PXC with their mCRFR1 counterparts did not rescue CRF or sauvagine radioligand binding or stimulation of cAMP accumulation. Replacement of residues 1−31 of mCRFR1 with their PTH1R counterparts resulted in a chimeric receptor, PEC, which had normal CRFR1 functional properties. In addition, a series of chimeras (F1PEC−F6PEC) were generated by replacement of the NT intercysteine residues of PEC with their PTH1R counterparts. Only F1PEC, F2PEC, and F3PEC showed detectable CRF and sauvagine radioligand binding. All of the PEC chimeras except F5PEC increased cAMP accumulation. These data indicate that the Cys68-Glu109 domain is important for binding and that the Cys87-Cys102 region plays an important role in CRFR1 activation.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>11170443</pmid><doi>10.1021/bi001758y</doi><tpages>9</tpages></addata></record> |
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subjects | Amino Acid Sequence Amphibian Proteins Animals COS Cells Cyclic AMP - metabolism Ligands Membrane Proteins - chemistry Membrane Proteins - genetics Membrane Proteins - metabolism Membrane Proteins - physiology Mice Mice, Inbred BALB C Molecular Sequence Data Mutagenesis, Insertional Peptide Fragments - chemistry Peptide Fragments - genetics Peptide Fragments - metabolism Peptide Fragments - physiology Peptide Hormones Peptides - pharmacology Protein Structure, Tertiary - genetics Proto-Oncogene Proteins c-myc - genetics Rats Receptor, Parathyroid Hormone, Type 1 Receptors, Corticotropin-Releasing Hormone - chemistry Receptors, Corticotropin-Releasing Hormone - genetics Receptors, Corticotropin-Releasing Hormone - metabolism Receptors, Corticotropin-Releasing Hormone - physiology Receptors, Parathyroid Hormone - chemistry Receptors, Parathyroid Hormone - genetics Receptors, Parathyroid Hormone - metabolism Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - metabolism Structure-Activity Relationship |
title | Juxtamembrane Region of the Amino Terminus of the Corticotropin Releasing Factor Receptor Type 1 Is Important for Ligand Interaction |
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