Abstract
After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G(s) activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated G(q) activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate beta-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.
Highlights
From the ‡Department of Pharmacology, Institute of Pharmacology and Toxicology, University of Wurzburg, D-97078 Wurzburg, Germany and the §Endocrine Research Unit, Veterans Affairs Medical Center, and the Departments of Medicine and Physiology, University of California, San Francisco, California 94121
Nitroxide spin-spin interaction experiments demonstrated that the molecular nature of the conformational change in rhodopsin associated with light-induced transducin (Gt) activation is linked to a relative movement and rotation of the cytoplasmic part of helix 3 away from the cytoplasmic part of helix 6 [11]
Using a similar zinc ion site engineering strategy, we have shown the importance of the relative movement between helices 3 and 6 for Gs activation in the 2-adrenergic receptor and the parathyroid hormone receptor (PTHR) [13]
Summary
Vol 276, No 36, Issue of September 7, pp. 33435–33443, 2001 Printed in U.S.A. Differential Conformational Requirements for Activation of G Proteins and the Regulatory Proteins Arrestin and G Protein-coupled Receptor Kinase in the G Protein-coupled Receptor for Parathyroid Hormone (PTH)/PTH-related Protein*. The mutants were indistinguishable from wild-type PTHR in the ability to translocate -arrestins/green fluorescent protein to the cell membrane and were not affected by sensitivity to Zn(II) These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Using a similar zinc ion site engineering strategy, we have shown the importance of the relative movement between helices 3 and 6 for Gs activation in the 2-adrenergic receptor and the parathyroid hormone receptor (PTHR) [13] These studies indicate that the relative motion between helices 3 and 6 is a common switch mechanism in GPCRs of class I (rhodopsinlike) and class II (PTHR-like) to activate G proteins. Our present data indicate that the conformational change leading to relative movement of helices 3 and 6 is required for Gq activation, but is not involved in the activation process of the regulatory proteins GRK-2 and arrestin. Taking advantage of the fact that in the presence of Zn(II), H401 and H402 signaling is blocked, we have directly examined the role of G protein activation in PTHR internalization
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