A proline-rich region of the third intracellular loop imparts phenotypic beta 1-versus beta 2-adrenergic receptor coupling and sequestration.
beta-Adrenergic receptor (beta AR) subtypes differ not only by characteristic ligand affinities but also in the manner and extent to which they mediate agonist-promoted events such as activation of adenylyl cyclase and receptor sequestration. We utilized mutagenesis and recombinant expression in Chinese hamster fibroblasts to examine the effect of an unusual proline-rich 24-amino acid sequence (PARPPSPSPSPVPAPAPPPGPPRP) present in the third intracellular loop of the beta 1AR, but not in the beta 2AR, on the aforementioned receptor-mediated events. Cells expressing the wild-type beta 2AR stimulated adenylyl cyclase in response to the agonist isoproterenol with an EC50 approximately 5-fold lower than that observed with the beta 1AR (0.53 +/- 0.14 versus 2.47 +/- 0.52 nM, p < 0.01). Deletion of the proline-rich sequence from the beta 1AR resulted in an improvement in isoproterenol-stimulated adenylyl cyclase to an EC50 value intermediate to that observed in the wild-type receptors (1.14 +/- 0.08 nM, p < 0.05 versus wild-type beta 1AR). In contrast, insertion of this sequence into the beta 2AR impaired its ability to mediate this process. Similar results were observed for receptor sequestration. Wild-type beta 1-and beta 2AR underwent maximal agonist-promoted sequestration of 25.9 +/- 4.0 and 60.0 +/- 3.3%, respectively. Deletion of the proline-rich region from the beta 1AR improved maximal sequestration to 43.0 +/- 2.7% (p < 0.01 versus wild-type beta 1AR), while insertion of the sequence into the beta 2AR impaired sequestration to 33.9 +/- 2.7% (p < 0.001 versus wild-type beta 2AR). We conclude that the distinct phenotypic patterns observed for these two agonist-promoted events in the beta 1AR and beta 2AR subtypes are partially due to the conformational effects of this proline-rich third intracellular loop sequence. Such regions, which are also found in some other G-protein-coupled receptors, may represent a general motif responsible for attenuating certain agonist-promoted receptor events.
Green
S A
SA
Department of Medicine (Pulmonary), University of Cincinnati Medical Center, Ohio 45267-0564.
Liggett
S B
SB
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D011392:Proline; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D011392:Proline; D000262:Adenylyl Cyclases