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Published online 11 February 2002. doi:10.1083/jcb.200110132
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© The Rockefeller University Press, 0021-9525/2002/2/665 $5.00
The Journal of Cell Biology, Volume 156, Number 4, February 18, 2002 665-676

Article

G protein–coupled receptor/arrestin3 modulation of the endocytic machinery



Francesca Santini, Ibragim Gaidarov and James H. Keen

Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107

Address correspondence to James H. Keen, Kimmel Cancer Institute and the Department of Microbiology and Immunology, Thomas Jefferson University, 233 South 10th St., BLSB/915, Philadelphia, PA 19107. Tel.: (215) 503-4624. Fax: (215) 503-0622. E-mail: jim.keen{at}mail.tju.edu

Nonvisual arrestins (arr) modulate G protein–coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing ß2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3–GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16°C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.

Key Words: clathrin; AP-2; endocytosis; actin; membrane skeleton


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