Robin Pals-Rylaarsdam

Agonist-Receptor-Arrestin, an Alternative Ternary Complex with High Agonist Affinity

The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the β2-adrenergic receptor (β2AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of β2AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated β2AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonist-receptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.

Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms.

The phenomenon of acute desensitization of G-protein-coupled receptors has been associated with several events, including receptor phosphorylation, loss of high affinity agonist binding, receptor:G-protein uncoupling, and receptor internalization. However, the biochemical events underlying these processes are not fully understood, and their contributions to the loss of signaling remain correlative. In addition, the nature of the kinases and the receptor domains which are involved in modulation of activity have only begun to be investigated. In order to directly measure the role of G-protein-coupled receptor kinases (GRKs) in the desensitization of the m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative allele of GRK2 was used to inhibit receptor phosphorylation by endogenous GRK activity in a human embryonic kidney cell line. The dominant-negative GRK2 reduced agonist-dependent phosphorylation of the m2 mAChR by 50% and prevented acute desensitization of the receptor as measured by the ability of the m2 mAChR to attenuate adenylyl cyclase activity. In contrast, the agonist-induced internalization of the m2 mAChR was unaffected by the GRK2 construct. Further evidence linking receptor phosphorylation to acute receptor desensitization was obtained when two deletions of the third intracellular loop were made which created m2 mAChRs that did not become phosphorylated in an agonist-dependent manner and did not desensitize. However, the mutant mAChRs retained the ability to internalize. These data provide the first direct evidence that GRK-mediated receptor phosphorylation is necessary for m2 mAChR desensitization; the likely sites of in vivo phosphorylation are in the central portion of the third intracellular loop (amino acids 282-323). These results also indicate that internalization of the m2 receptor is not a key event in desensitization and is mediated by mechanisms distinct from GRK phosphorylation of the receptor.

ARRESTIN-INDEPENDENT INTERNALIZATION OF THE M1, M3, AND M4 SUBTYPES OF MUSCARINIC CHOLINERGIC RECEPTORS

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682–23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the β2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.

Two Homologous Phosphorylation Domains Differentially Contribute to Desensitization and Internalization of the m2 Muscarinic Acetylcholine Receptor

Short term exposure of m2 muscarinic acetylcholine receptors (m2 mAChRs) to agonist causes a rapid phosphorylation of the activated receptors, followed by a profound loss in the ability of the m2 mAChR to activate its signaling pathways. We have used site-directed mutagenesis to identify two clusters of Ser/Thr residues in the third intracellular loop of the m2 mAChR that can serve as redundant targets for agonist-dependent phosphorylation. Mutation of both clusters of Ser/Thr residues to alanines abolished agonist-dependent phosphorylation, while wild-type levels of m2 mAChR phosphorylation were observed in mutant receptors with only one or the other cluster mutated. However, the functional effects of phosphorylation of these two “redundant” clusters were not equivalent. No receptor desensitization was observed in an m2 mAChR with residues Thr307–Ser311 mutated to alanine residues. In contrast, mutation of the other Ser/Thr cluster, residues Ser286–Ser290, to alanines produced a receptor that continued to desensitize. Internalization of the m2 mAChR was promoted by phosphorylation of either cluster, suggesting that distinct mechanisms with unique structural requirements act downstream of m2 mAChR phosphorylation to mediate receptor desensitization and receptor internalization.

Chapter 15 The role of G-protein coupled receptor kinases in the regulation of muscarinic cholinergic receptors

Publisher Summary This chapter focuses exclusively on molecular events underlying homologous desensitization of muscarinic acetylcholine receptors (mAChR) by a unique family of protein kinases that appear to specifically recognize GPRs. Other protein kinases, such as protein kinase C, may also be involved in mAChR desensitization. The term “desensitization”, when applied to the GPRs, actually refers to a collection of events that may be independently regulated, and not necessarily sequential. The hallmarks of desensitization of the GPRs include uncoupling of the receptors from G-proteins and/or loss of high affinity agonist binding. These events occur rapidly and appear to involve phosphorylation of the GPRs by specific and/or generic protein kinases. This phosphorylation induced uncoupling is proposed to play a physiological role in terminating signalling in the sensory receptor systems. The chapter identifies the kinases that phosphorylate the mAChRs in vivo employs a system, whereby it attempts to take apart the system and reconstitutes agonist-dependent phosphorylation and desensitization of the receptors in vitro . For this system, the chapter purifies chick heart or recombinant human m2 mAChRs and reconstitutes the receptors into phospholipid vesicles.

A novel double mutation in the luteinizing hormone receptor in a kindred with familial Leydig cell hypoplasia and male pseudohermaphroditism.

We report a novel mutant of the luteinizing hormone receptor (LHR) in a case of familial Leydig cell hypoplasia and pseudohermaphrotidism. The proband was homozygous for two missense mutations, T1121C and C1175T, causing substitutions I374T and T392I. The molecular effects of the mutations were investigated by heterologous expression of the WT LHR, the double mutant LHR, or receptors with either the I374T or the T392I mutation, and measuring hormone binding and cAMP signaling. All mutant LHRs exhibited severe defects, including loss of ligand binding and cAMP production. Immunoblots showed little difference in protein levels between the WT and mutant receptors.

Molecular events associated with the regulation of signaling by M2 muscarinic receptors.

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.