Effects of mutations in the highly conserved DRY motif on binding affinity, expression, and G-protein recruitment of the human angiotensin II type-2 receptor.
The signaling pathways for the seven transmembrane G-protein coupled angiotensin II receptors (AT(1) and AT(2)) are just beginning to be understood. While these receptors play an important role in the development and differentiation of many tissues, including the cardiovascular and central nervous systems, information about amino acid motifs involved in angiotensin II-mediated signaling is only available for the AT(1) receptor subtype. In the present study, we mutated the conserved DRY(141-143) motif in the AT(2) receptor, which is thought to be involved in G-protein recruitment. Expression of wild type and mutant receptors in CHO-K1 cell plasma membranes was confirmed using radioligand binding analyses. Our findings indicate a significant change in the binding affinities (kD) and capacities (B(max)) of the mutant receptors relative to wild type. Alanine substitutions of D(141) and DRY(141-143) resulted in a significant decrease of binding affinity for both Sar(1)Ile(8)-angiotensin II (SarIle-Ang II) (mixed agonist/antagonist) and angiotensin II (agonist). The binding affinities following alanine substitutions of R(142) and Y(143) were not significantly different from wild type receptor. Interestingly, the R(142)-A and Y(143)-A mutants revealed a significant decrease in binding levels from wild type with SarIle-Ang II, but not angiotensin II. The effect of GTPgammaS on angiotensin II binding affinity between wild type and mutant receptors was similarly significant. The D(141)-A, Y(143)-A, and DRY(141-143)-AAA mutant receptors showed a marked decrease in GTPgammaS-induced angiotensin II affinity shift. The R(142)-A GTPgammaS binding affinity shift was not different from the wild type receptor. Our results support the hypothesis that the DRY motif plays a significant role in the binding affinity, structural stability and G-protein recruiting of the AT(2) receptor.
Moore
Steven A
SA
Department of Cell and Structural Biology, University of Illinois, B107 Chemical and Life Science Building, 601 South Goodwin Avenue, Urbana, IL 61801, USA.
Patel
Anjali S
AS
Huang
Nancy
N
Lavin
Bridget C
BC
Grammatopoulos
Tom N
TN
Andres
Robert D
RD
Weyhenmeyer
James A
JA
D020816:Amino Acid Motifs; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011869:Radioligand Assay; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015290:Second Messenger Systems / Q000502:physiology*
D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins