Differential regulation of G protein α‐subunit GTPase activity by peptides derived from the third cytoplasmic loop of the α₂‐adrenergic receptor

Abstract

The effect of peptides homologous to segments of a G protein-coupled receptor on the GTPase activity of recombinant Goa (rGoa) and G~a (rG~a) has been tested. These peptides contain overlapping sequences spanning from amino acid 212 of the putative fifth transmembrane domain to amino acid 229 of the third cytoplasmic loop of the oz2 adrenergic receptor. Interestingly, two peptides (comprising residues 212-227 and 214-227) strongly inhibit the basal GTPase activity of both rGo~ and rG~. Instead, a C-terminally extended peptide (residues 216-229) stimulates rGo~ but slightly inhibits rG~a. Circular dichroism spectroscopy of the peptides reveals that an a helical structure is more easily inducible in the inhibitory ones. These findings constitute an example of peptides representing cytoplasmic receptor sequences that differentially modulate the GTPase activity of recombinant G protein a-subunits.

Structure of Two Fragments of the Third Cytoplasmic Loop of the Rat Angiotensin II AT1A Receptor

“…These considerations, and the results obtained on that subject for rhodopsin (7)(8)(9)(10)(11), for the ␣-and ␤-adrenergic receptors (12)(13)(14), and for the parathormone receptor (15,16) have prompted us to undertake a study on some functionally relevant cytoplasmic domains of the angiotensin II AT 1A receptor, mainly seeking to describe the structural and dynamic properties of the receptor surface that regulate its interaction with the various G-proteins.…”

Is Promiscuity of G Protein Interaction an Issue in the Classification of Receptors?

“…For example, in the case of the metabotropic glutamate receptors, strong evidence indicates major roles for both the second intracellular loop and the C terminal tail in the regulation of G protein contacts 29 and as the splice variation within the EP3 receptor is restricted entirely to the C terminal tail this must be a key site of interaction for this receptor. The use of peptides derived from the end of the fifth transmembrane element and the early part of the third intracellular loop of the α 2A ‐adrenoceptor have also recently provided evidence to indicate not only that receptors may show promiscuity of coupling to G proteins 30 but also that certain receptors may have the capacity to inhibit the GTPase of certain G proteins. Peptides corresponding to amino acids 212–227 and 214–227 of the α 2A ‐adrenoceptor were able to inhibit the basal GTPase activity of both recombinant G o α and G s α 30 .…”

“…The use of peptides derived from the end of the fifth transmembrane element and the early part of the third intracellular loop of the α 2A ‐adrenoceptor have also recently provided evidence to indicate not only that receptors may show promiscuity of coupling to G proteins 30 but also that certain receptors may have the capacity to inhibit the GTPase of certain G proteins. Peptides corresponding to amino acids 212–227 and 214–227 of the α 2A ‐adrenoceptor were able to inhibit the basal GTPase activity of both recombinant G o α and G s α 30 . As peptides derived from very similar regions of this receptor can cause stimulation of G protein GTPase activity, 31 such results appear to indicate that rather subtle changes in receptor structure and conformation may be sufficient to change the way in which the cellular G protein population may be regulated by receptor occupation by agonists.…”