Agonist-regulated Interaction between alpha2-adrenergic receptors and spinophilin.
Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical alpha2A-adrenergic receptor (alpha2A AR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether alpha(2)AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the alpha2A AR (Val(217)-Ala(377)), alpha2BAR (Lys(210)-Trp(354)), and alpha2CAR (Arg(248)-Val(363)) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169-255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because alpha2A AR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to alpha2 AR localization but also to agonist modulation of alpha2AR signaling.
Richman
J G
JG
Departments of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Brady
A E
AE
Wang
Q
Q
Hensel
J L
JL
Colbran
R J
RJ
Limbird
L E
LE
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D005982:Glutathione Transferase / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000099273:Neurabins
D000316:Adrenergic alpha-Agonists; D005982:Glutathione Transferase; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D000099273:Neurabins