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The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of \u03b1<jats:sub>1<\/jats:sub>\u2010ARs. Here, using thermostabilized, purified, \u03b1<jats:sub>1A<\/jats:sub>\u2010 and \u03b1<jats:sub>1B<\/jats:sub>\u2010ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized \u03b1<jats:sub>1A<\/jats:sub>\u2010 and \u03b1<jats:sub>1B<\/jats:sub>\u2010ARs. Similarly, in cell\u2010based assays expressing unmodified \u03b1<jats:sub>1A<\/jats:sub>\u2010 and \u03b1<jats:sub>1B<\/jats:sub>\u2010ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist\u2010stimulated Ca<jats:sup>2+<\/jats:sup> release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by \u03b1<jats:sub>1A<\/jats:sub>\u2010 and \u03b1<jats:sub>1B<\/jats:sub>\u2010ARs; however, this was shown to be caused by off\u2010target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.<\/jats:p>","DOI":"10.1002\/prp2.455","type":"journal-article","created":{"date-parts":[[2018,12,27]],"date-time":"2018-12-27T07:39:19Z","timestamp":1545896359000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Diazepam is not a direct allosteric modulator of \u03b1<sub>1<\/sub>\u2010adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase\u20104"],"prefix":"10.1002","volume":"7","author":[{"given":"Lisa M.","family":"Williams","sequence":"first","affiliation":[{"name":"The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoji","family":"He","sequence":"additional","affiliation":[{"name":"The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia"},{"name":"School of Chemistry and Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tasneem M.","family":"Vaid","sequence":"additional","affiliation":[{"name":"The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia"},{"name":"Department of Biochemistry and Molecular Biology University of Melbourne Parkville Vic Australia"},{"name":"The Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alaa","family":"Abdul\u2010Ridha","sequence":"additional","affiliation":[{"name":"The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alice R.","family":"Whitehead","sequence":"additional","affiliation":[{"name":"The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0323-449X","authenticated-orcid":false,"given":"Paul R.","family":"Gooley","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology University of Melbourne Parkville Vic Australia"},{"name":"The Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6301-861X","authenticated-orcid":false,"given":"Ross A.D.","family":"Bathgate","sequence":"additional","affiliation":[{"name":"The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia"},{"name":"Department of Biochemistry and Molecular Biology University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6341-4364","authenticated-orcid":false,"given":"Spencer J.","family":"Williams","sequence":"additional","affiliation":[{"name":"School of Chemistry and Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville Vic Australia"},{"name":"The Bio21 Molecular Science and Biotechnology Institute University of Melbourne Parkville Vic Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6332-2793","authenticated-orcid":false,"given":"Daniel J.","family":"Scott","sequence":"additional","affiliation":[{"name":"The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia"},{"name":"Department of Biochemistry and Molecular Biology University of Melbourne 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