UC San Diego

In this dissertation, I identify the covalent modification of caveolin-3 with small-ubiquitin-like modifier (SUMO) proteins and its interactions with components of the sumoylation machinery. Caveolins are the principal protein components of caveolae membrane microdomains and function as scaffolds that compartmentalize and regulate multiple signaling proteins, their downstream effectors and associated proteins. Caveolin-3 is a muscle-specific caveolin family member that is highly expressed in skeletal muscle and cardiac myocytes. Beta 1- and beta 2- adrenergic receptors ([beta]ARs) use distinct pathways to regulate contraction in cardiac myocytes. Caveolar localization and interaction with caveolin-3 is required for signaling by the [beta]₂AR but not the [beta]₁AR. [beta]₂ARs and [beta]₁ARs also desensitize through distinct pathways in response to agonist stimulation. Evidence presented here suggests that sumoylation of caveolin-3 is involved in the regulation of agonist- induced desensitization of [beta]₂ARs. Sumoylation is a highly dynamic and reversible posttranslational modification that regulates substrates involved in many aspects of cellular function. SUMO E1, E2 and E3 enzymes catalyze the formation of covalent, isopeptide bonds with lysine side chains on target substrates while SUMO- specific proteases (SENPs) remove SUMO proteins from substrates. Experiments in this thesis show that caveolin- 3 is covalently modified by SUMO and poly-SUMO chains in multiple cell types. In vitro and in vivo sumoylation assays demonstrate that modification of caveolin-3 by poly -SUMO-3 chains is enhanced by co-expression of the SUMO E3 ligase PIASy in a dose-dependent manner. Co-expression of SENP1 or SENP2 dramatically reduced modification of caveolin-3 by SUMO-3. Site-directed mutagenesis was used to identify the preferred site of sumoylation and create a sumoylation-deficient mutant to probe the biological function of SUMO modification. Co-expression of caveolin-3 or the sumoylation-deficient mutant with [beta]ARs showed differential effects on the stability of [beta]₂AR, but not [beta]₁AR, expression levels in the presence of prolonged agonist stimulation. This effect on [beta]₂ARs was attenuated by the [beta]AR antagonist (-)-propranolol. Taken together, the data in this dissertation identify sumoylation as a novel mechanism for the regulation of caveolin and its interactions with its signaling partners, including effects on the agonist-induced desensitization of [beta]₂ARs